HK1232553B - Method of modifying isoelectric point of antibody via amino acid substitution in cdr - Google Patents

Description

Method for changing the isoelectric point of antibodies by using CDR amino acid substitution

The present application is a divisional application of an invention patent application filed on September 26, 2008, with application number 200880118639.2 and the invention name being “Method for changing the isoelectric point of an antibody by utilizing amino acid substitution of CDR”.

Technical Field

The present invention relates to a method for modifying the isoelectric point of an antibody while maintaining its antigen-binding activity by using amino acid substitutions in its CDRs; a method for controlling the plasma pharmacokinetics (hemodynamics) of an antibody; and a pharmaceutical composition containing an antibody with a modified isoelectric point as an active ingredient and a method for producing the same.

The present invention also relates to methods for controlling the plasma half-life of anti-IL-6 receptor antibodies, anti-glypican 3 antibodies, and anti-IL-31 receptor antibodies by modifying amino acid residues exposed on the surface of their CDR regions; antibodies (anti-IL-6 receptor antibodies, anti-glypican 3 antibodies, and anti-IL-31 receptor antibodies) whose plasma half-life is controlled by modification of amino acid residues; pharmaceutical compositions containing the antibodies as active ingredients; and methods for producing these pharmaceutical compositions.

The present invention also relates to a pharmaceutical composition containing an anti-IL-6 receptor antibody as an active ingredient and a method for producing the same.

Background Art

Antibody has a long half-life in plasma and few side effects, so it is paid attention to as medicine. Wherein, there are multiple IgG type antibody drugs on the market, and multiple antibody drugs are also under development (non-patent literature 1, non-patent literature 2). The antibody drugs on the market now are basically chimeric antibodies, humanized antibodies, and people's antibodies, which will be improved by humanized antibodies or people's antibodies, improve their drug effect, convenience, cost, so as to have the multiple antibody drugs of more excellent characteristics under development. As the technology that can be applicable to these antibody drugs, people have developed various technologies, and someone has reported the technology that improves effector function, antigen binding ability, pharmacokinetics, stability or reduces the technology of immunogenicity risk etc. As the method for enhancing drug effect or reducing dosage, someone has reported the technology (non-patent literature 3,4) that enhances antibody-dependent cellular toxicity (ADCC activity) or complement-dependent cytotoxicity (CDC activity) by replacing the amino acid in IgG antibody Fc district. Furthermore, affinity maturation has been reported as a technique for improving antigen-binding and antigen-neutralizing abilities (Non-Patent Document 5). This technique introduces amino acid mutations into the CDR regions of the variable region to enhance antigen-binding activity.

The problems faced by current antibody drugs are: high manufacturing costs caused by a large amount of protein administration. Regarding the mode of administration, when it is a chronic autoimmune disease, a subcutaneous administration preparation is preferred, but usually a subcutaneous administration preparation must be a high concentration preparation. When it is an IgG type antibody preparation, from the perspective of stability, it is generally believed that a preparation of about 100 mg/mL is the limit (non-patent literature 6). In order to be able to exert a sustained therapeutic effect and extend the plasma half-life of the antibody, the amount of protein administered can be reduced, and subcutaneous administration can be performed at a longer dosing interval, which can provide an antibody drug with excellent characteristics that is low in cost and convenient.

The half-life in the plasma of antibodies is long and is closely related to FcRn. Regarding the difference in half-life in the plasma between antibody isotypes, the half-life in the plasma of known IgG1 and IgG2 is the longest, while the half-life in the plasma of IgG3 and IgG4 is shorter than that of IgG1 and IgG2 (non-patent literature 7). As a method for further extending the half-life in the plasma of IgG1 and IgG2 antibodies with excellent half-life in plasma, it has been reported that amino acid substitutions in the constant region that enhance the binding to FcRn (non-patent literature 8, 9, 10) have been reported. However, from the perspective of immunogenicity, there are problems with introducing artificial amino acid mutations in the constant region. In contrast, recently, it has been reported that a method for improving the pharmacokinetics of antibodies by introducing mutations in the amino acids of the antibody variable region (patent literature 1) has been reported.

According to Patent Document 1, the pharmacokinetics of IgG can be controlled by altering the isoelectric point. Furthermore, by introducing amino acid substitutions into the framework of an antibody's variable region, the isoelectric point of an antibody can be lowered and its plasma half-life extended without reducing its antigen-binding activity. Specifically, for example, by introducing amino acid substitutions into Kabat-numbered sequences H10, H12, H23, H39, H43, and H105, the isoelectric point of an antibody can be lowered without reducing its antigen-binding activity. Furthermore, amino acid mutations can be introduced into other framework sequences without reducing binding activity. However, some believe that introducing amino acid substitutions into the framework sequence alone is not sufficient to significantly lower the isoelectric point. This is because the framework sequence after amino acid substitutions is typically based on human antibody sequences to reduce immunogenicity. However, human antibody framework sequences are highly conserved and less diverse, resulting in limited freedom for amino acid substitutions. Therefore, if introducing amino acid substitutions into the framework alone does not sufficiently lower the isoelectric point of an antibody, further lowering the isoelectric point is difficult.

On the other hand, CDR sequences have a wide variety due to somatic mutations, and have the variety for obtaining antigen-binding, so compared with framework, the degree of freedom of amino acid substitution is significantly larger. However, known CDR sequences are the most important factor for exerting strong antigen-binding activity, and usually amino acid substitutions of CDR sequences can affect the antigen-binding activity of antibody. Therefore, it is difficult to utilize amino acid substitutions of CDR sequences to reduce the isoelectric point of antibody when the antigen-binding activity of antibody is not significantly reduced. In addition, because CDR sequences vary greatly according to antigen species, it is believed that, when not relying on antibody species, not significantly reducing the antigen-binding activity of antibody, it is extremely difficult to replace the amino acid of the CDR sequence of antibody. In fact, this situation is not difficult to infer from a plurality of findings shown below.

When humanizing antibodies derived from non-human animal species, CDR grafting is typically used, in which the CDR sequences of the non-human animal species are transplanted into human framework sequences. When the humanized antibody obtained by CDR grafting does not have the same binding activity as the chimeric antibody, the binding activity can be restored by substituting a portion of the amino acids in the framework sequence that determines the CDR structure with the framework sequence of the antibody from the non-human animal species from which the antibody is derived (Non-Patent Document 11). As mentioned above, the sequence and structure of the CDRs are extremely important for the antigen-binding activity and specificity of the antibody. It is well known that changes in the CDR residues of an antibody, such as isomerization of aspartic acid residues, deamidation of asparagine residues, and oxidation of methionine residues in the antibody CDRs, can weaken the antigen-binding activity of the antibody (Non-Patent Document 12), which also shows that the CDR sequence is extremely important for the antigen-binding activity of the antibody. In addition, it has been reported that the introduction of amino acid substitutions into the CDR2 sequence of the antibody H chain often significantly reduces the antigen-binding activity and reduces the expression level of the antibody (Non-Patent Documents 13-15). It has been clarified that, in particular, when amino acid substitutions are introduced into H51, the expression level of the antibody is significantly reduced (non-patent literature 16). In addition, when mutations are introduced into the CDR3 sequence of the antibody H chain, the antigen binding activity is often greatly weakened (non-patent literature 17, 18). When alanine scanning of the antibody CDR sequence is performed, the antigen binding activity of the antibody is often greatly weakened by replacing the amino acids present in the CDR with alanine (non-patent literature 19-23). It is believed that when substituted with alanine, the effect on the antigen binding activity varies depending on the type of antibody. That is, it is generally believed that by replacing the amino acids in the antibody CDR sequence, its antigen binding activity will be weakened. To date, there has been no report on amino acid substitution sites that do not depend on the type of antibody and do not significantly reduce the antigen binding activity of the antibody.

In antibody engineering, which aims to develop antibody molecules with superior properties, amino acid substitutions introduced into antibody CDR sequences are almost always targeted for affinity maturation. Affinity maturation generally refers to a method in which an antibody library with randomized CDR sequences is displayed on phage or ribosomes, and then panned against antigen to obtain antibodies with further enhanced antigen-binding activity. This method can be used to identify amino acid substitutions in antibody CDR sequences that enhance antigen-binding activity (Non-Patent Documents 5, 24-26). However, the amino acid substitutions that enhance antigen-binding activity obtained by this method vary depending on the antibody species, and thus, to date, there have been no reports on amino acid substitution sites in CDR sequences that enhance antigen-binding activity independently of the antibody species. In addition to affinity maturation, a method has been reported for enhancing antibody expression in mammalian cells by substituting amino acids at specific sites in the CDR sequence (Patent Document 2). According to Patent Document 2, by substituting amino acids at specific sites in the CDR sequence with specific sequences, antibody expression in mammalian cells can be enhanced independently of the antibody species. Some people have also reported that de-immunization is performed to avoid T cell epitopes present in the antibody CDR sequence in order to reduce the immunogenicity of the antibody. However, to date, there has been no report on an amino acid substitution method for removing T cell epitopes present in the CDR sequence that is independent of the antibody type and does not reduce the binding activity of the antibody (Non-Patent Literature 27, 28).

As mentioned above, since the CDR sequence of an antibody is closely related to its antigen-binding activity, amino acid substitutions in the CDR sequence generally reduce its binding activity. The effect of amino acid substitutions in the CDR sequence on its antigen-binding activity varies depending on the type of antibody. Patent Document 1 provides an example of controlling the isoelectric point using amino acid substitutions in the CDR, but it is believed that depending on the type of antibody, it is possible to reduce the antigen-binding activity. In addition, some have reported methods for increasing antibody expression using common amino acid substitutions, but to date, there have been no reports of increasing the antigen-binding activity of an antibody, nor have there been reports of methods for removing T cell epitopes without significantly reducing the antigen-binding activity of an antibody. Furthermore, there are no reports at all on CDR sequences of antibodies that can undergo amino acid substitutions without significantly reducing the antigen-binding activity of an antibody, regardless of the type of antibody.

It should be noted that the prior art documents of the present invention are as follows.

Non-patent document 1: Monoclonal antibody successes in the clinic, Janice M Reichert, Clark J Rosensweig, Laura B Faden & Matthew C Dewitz. Nature Biotechnology 23: 1073-1078 (2005).

Non-patent document 2: Pavlou AK, Belsey MJ. The therapeutic antibodies market to 2008. Eur J Pharm Biopharm. 2005 Apr; 59(3): 389-96.

Non-patent document 3: Presta LG. Engineering of therapeutic antibodies to minimize immunogenicity and optimize function. Adv Drug Deliv Rev. 2006 Aug 7; 58(5-6): 640-56.

Non-patent literature 4: Kim SJ, Park Y, Hong HJ. Antibody engineering for the development of therapeutic antibodies. Mol Cells. 2005 Aug 31; 20(1): 17-29 Review.

Non-patent document 5: Fujii I. Antibody affinity maturation by random mutagenesis. Methods Mol Biol. 2004; 248: 345-59.

Non-patent literature 6: Shire SJ, Shahrokh Z, Liu J. Challenges in the development of high protein concentration formulations. J Pharm Sci. 2004 Jun; 93(6): 1390-402.

Non-patent literature 7: Salfeld JG. Isotype selection in antibody engineering. Nat Biotechnol. 2007 Dec; 25(12): 1369-72.

Non-patent literature 8: Hinton PR, Johlfs MG, Xiong JM, Hanestad K, Ong KC, Bullock C, Keller S, Tang MT, Tso JY, Vasquez M, Tsurushita N. Engineered human IgGantibodies with longer serum half-lives in primates. J Biol Chem. 2004 Feb 20;279(8):6213-6.

Non-patent literature 9: Hinton PR, Xiong JM, Johlfs MG, Tang MT, Keller S, Tsurushita N. An engineered human IgG1 antibody with longer serum half-life. JImmunol. 2006 Jan 1; 176(1): 346-56.

Non-patent literature 10: Ghetie V, Popov S, Borvak J, Radu C, Matesoi D, Medesan C, Ober RJ, Ward ES. Increasing the serum persistence of an IgG fragment by randommutagenesis. Nat Biotechnol. 1997 Jul; 15(7): 637-40.

Non-patent literature 11: Almagro JC, Fransson J. Humanization of antibodies. Front Biosci. 2008 Jan 1; 13: 1619-33.

Non-patent literature 12: Liu H, Gaza-Bulseco G, Faldu D, Chumsae C, Sun J. Heterogeneity of monoclonal antibodies. J Pharm Sci. 2008 Jul; 97(7): 2426-47.

Non-patent literature 13: Chen C, Roberts VA, Rittenberg MB. Generation and analysis of random point mutations in an antibody CDR2 sequence: many mutatedantibodies lose their ability to bind antigen. J Exp Med. 1992 Sep 1; 176(3):855-66.

Non-patent literature 14: Chen C, Martin TM, Stevens S, Rittenberg MB. Defective secretion of an immunoglobulin caused by mutations in the heavy chaincomplementarity determining region 2. J Exp Med. 1994 Aug1; 180(2):577-86.

Non-patent literature 15: Wiens GD, Heldwein KA, Stenzel-Poore MP, RittenbergMB. Somatic mutation in VH complementarity-determining region 2 and framework region 2: differential effects on antigen binding and Ig secretion. JImmunol. 1997 Aug 1; 159(3): 1293-302.

Non-patent literature 16: Wiens GD, Lekkerkerker A, Veltman I, Rittenberg MB. Mutation of a single conserved residue in VH complementarity-determining region 2results in a severe Ig secretion defect. J Immunol. 2001 Aug 15; 167(4): 2179-86.

Non-patent literature 17: Zwick MB, Komori HK, Stanfield RL, Church S, Wang M, Parren PW, Kunert R, Katinger H, Wilson IA, Burton DR. The long third complementarity-determining region of the heavy chain is important in the activity of the broadly neutralizing anti-human immunodeficiency virus type 1antibody 2F5.JVirol.2004 Mar;78(6):3155-61.

Non-patent literature 18: Komissarov AA, Marchbank MT, Calcutt MJ, Quinn TP, DeutscherSL. Site-specific mutagenesis of a recombinant anti-single-stranded DNAFab. Role of heavy chain complementarity-determining region 3 residues inantigen interaction. J Biol Chem. 1997 Oct 24; 272(43):26864-70.

Non-patent literature 19: Gerstner RB, Carter P, Lowman HB. Sequence plasticity in the antigen-binding site of a therapeutic anti-HER2 antibody. J Mol Biol. 2002Aug 30; 321(5):851-62.

Non-patent literature 20: Vajdos FF, Adams CW, Breece TN, Presta LG, de Vos AM, SidhuSS. Comprehensive functional maps of the antigen-binding site of an anti-ErbB2antibody obtained with shotgun scanning mutagenesis. J Mol Biol. 2002 Jul 5;320(2):415-28.

Non-patent literature 21: Pons J, Rajpal A, Kirsch JF. Energetic analysis of anantigen/antibody interface: alanine scanning mutagenesis and double mutantcycles on the HyHEL-10/lysozyme interaction. Potein Sci. 1999 May; 8(5): 958-68.

Non-patent literature 22: Leong SR, DeForge L, Presta L, Gonzalez T, Fan A, Reichert M, Chuntharapai A, Kim KJ, Tumas DB, Lee WP, Gribling P, Snedecor B, Chen H, Hsei V, Schoenhoff M, Hale V, Deveney J, Koumenis I, Shahrokh Z, McKay P, Galan W, Wagner B, Narindray D, Hebert C, Zapata G. Adapting pharmacokinetic properties of ahumanized anti-interleukin-8antibody for therapeutic applications using site-specific pegylation. Cytokine. 2001 Nov 7; 16(3): 106-19.

Non-patent literature 23: Xiang J, Srivamadan M, Rajala R, Jia Z. Study of B72.3 combining sites by molecular modeling and site-directed mutagenesis. ProteinEng. 2000 May; 13(5): 339-44.

Non-patent literature 24: Rothe A, Hosse RJ, Power BE. Ribosome display for improved biotherapeutic molecules. Expert Opin Biol Ther. 2006 Feb; 6(2): 177-87.

Non-patent literature 25: Schmitz U, Versmold A, Kaufmann P, Frank HG. Phage display: amolecular tool for the generation of antibodies--a review. Placenta. 2000 Mar-Apr; 21 Suppl A: S106-12.

Non-patent literature 26: Rajpal A, Beyaz N, Haber L, Cappuccilli G, Yee H, Bhatt RR, Takeuchi T, Lerner RA, Crea R. A general method for greatly improving the affinity of antibodies by using combinatorial libraries. Proc Natl Acad Sci US A. 2005 Jun 14; 102(24): 8466-71.

Non-patent document 27: De Groot AS, Knopp PM, Martin W. De-immunization of therapeutic proteins by T-cell epitope modification. Dev Biol (Basel). (2005) 122: 171-94.

Non-Patent Document 28:

http://www.algonomics.com/proteinengineering/tripole_applications.php.

Patent Document 1: WO/2007/114319

Patent Document 2: US/2006/0019342

Summary of the Invention

Problems to be solved by the invention

The present invention is made in view of the above-mentioned situation, and its purpose is to provide: a method for changing the isoelectric point of a polypeptide while maintaining its antigen-binding activity, wherein the polypeptide comprises an antibody variable region; a method for controlling the plasma half-life of an antibody; a pharmaceutical composition containing an antibody with a controlled plasma half-life as an active ingredient; and a method for producing the antibody and the pharmaceutical composition containing the antibody as an active ingredient.

The present invention also aims to provide: a method for controlling the plasma half-life of anti-IL-6 receptor antibodies, anti-glypican 3 antibodies, and anti-IL-31 receptor antibodies by modifying amino acid residues exposed on the surface of the CDR regions of the above antibodies; anti-IL-6 receptor antibodies, anti-glypican 3 antibodies, and anti-IL-31 receptor antibodies whose plasma half-life is controlled by modifying amino acid residues; a method for producing the above antibodies; and a pharmaceutical composition containing the above antibodies as an active ingredient.

Furthermore, the present invention aims to provide pharmaceutical compositions comprising second-generation molecules superior to tocilizumab, which are derived by modifying the amino acid sequences of the variable and constant regions of tocilizumab, a humanized anti-IL-6 receptor IgG1 antibody, to enhance antigen neutralization and improve plasma retention, thereby reducing dosing frequency, maintaining sustained therapeutic effects, and improving immunogenicity, safety, and physicochemical properties, as well as methods for producing such pharmaceutical compositions.

Solutions to Problems

The present inventors have conducted in-depth research on methods for changing the isoelectric point of a polypeptide comprising an antibody variable region while maintaining the antigen-binding activity of the variable region. As a result, the present inventors have discovered specific positions on the CDR amino acid sequence that can change the isoelectric point of the CDR while maintaining the antigen-binding activity of the variable region among the amino acid residues constituting the complementarity determining region (CDR) of the antibody variable region. The present inventors have also discovered that by controlling the isoelectric point of a polypeptide comprising an antibody variable region, the plasma half-life of the polypeptide can be controlled; and by utilizing the difference in isoelectric point, polypeptides comprising an antibody variable region composed of heteropolymers can be efficiently produced. Specifically, the present inventors have determined, among the amino acid residues in the amino acid sequence constituting the antibody variable region, positions on the specific CDR amino acid sequence that can regulate the surface charge of the antibody molecule without affecting the functions and structures of the antibody, such as the antigen-binding activity of the antibody variable region. Furthermore, the present inventors have confirmed that by regulating the surface charge of the antibody, the isoelectric point can be altered, and the plasma half-life of polypeptides comprising the antibody variable regions can be controlled. Thus, antibodies with controlled plasma half-lives actually retain antigen-binding activity. Furthermore, the present inventors have confirmed that by controlling the plasma half-life of antibodies, the tumor growth inhibition effect of cytotoxic antibodies, such as antibodies, on cancer cells is enhanced, thereby completing the present invention. Furthermore, the present inventors have confirmed that by regulating the charge of the CDRs, the isoelectric point can be altered, allowing the isolation and purification of heterodimers comprising antibodies that bind to two or more different antigens.

Furthermore, the present inventors conducted intensive research to develop a second-generation molecule superior to tocilizumab. This second-generation molecule, which is superior to tocilizumab, is based on amino acid sequence modifications to the variable and constant regions of tocilizumab, a first-generation humanized anti-IL-6 receptor IgG1 antibody, to enhance efficacy and improve plasma retention, thereby reducing dosing frequency and maintaining therapeutic effects. Furthermore, the inventors discovered multiple CDR mutations in the variable region of tocilizumab that enhance antigen-binding ability (affinity). Combinations of these mutations significantly improved affinity. Furthermore, the present inventors successfully improved plasma retention by introducing modifications that lower the isoelectric point of the variable region sequence. Furthermore, the present inventors successfully reduced the risk of immunogenicity by reducing the number of T-cell epitope peptides predicted in silico in the mouse-derived sequences and variable regions remaining in the tocilizumab framework. Furthermore, the present inventors successfully improved stability at high concentrations. Furthermore, the present inventors have successfully discovered a novel constant region sequence in the constant region of tocilizumab that minimizes the appearance of novel T-cell epitope peptides, while also preventing Fcγ receptor binding and improving stability under acidic conditions, heterogeneity derived from hinge region disulfide bonds, heterogeneity derived from the H chain C-terminus, and stability in high-concentration formulations. By combining these modifications in the CDR region amino acid sequence, the variable region amino acid sequence, and the constant region amino acid sequence, they successfully developed a second-generation molecule superior to tocilizumab.

More specifically, the present invention provides the following [1] to [44].

[1] A method for altering the isoelectric point of a polypeptide, wherein the method is to alter the isoelectric point of a polypeptide comprising an antibody variable region while maintaining the antigen-binding activity of the variable region, the method comprising: altering the charge of at least one amino acid residue that can be exposed on the surface of a complementarity determining region (CDR) of the polypeptide;

[2] The method of [1], wherein the polypeptide comprising an antibody variable region further comprises an FcRn binding region;

[3] The method described in [1], wherein the polypeptide comprising an antibody variable region is an IgG antibody;

[4] The method of [1], wherein the polypeptide comprising an antibody variable region is a chimeric antibody, a humanized antibody, or a human antibody;

[5] The method of [1], wherein the polypeptide comprising an antibody variable region is a multispecific polypeptide that binds to at least two antigens;

[6] The method of [1], wherein the change in the charge of the amino acid residue is an amino acid substitution;

[7] The method of [1], wherein the change in the charge of the amino acid residue is a change that causes the theoretical isoelectric point to change by 1.0 or more;

[8][1] The method according to claim 1, wherein the amino acid residue capable of being exposed on the surface of the CDR region is at least one amino acid residue selected from the group consisting of amino acid residues at positions 31, 61, 62, 64 and 65 according to Kabat numbering in the heavy chain variable region or amino acid residues at positions 24, 27, 53, 54 and 55 according to Kabat numbering in the light chain variable region;

[9] A polypeptide comprising an antibody variable region whose isoelectric point has been altered, obtained by the method of any one of [1] to [8];

[10] A method for controlling the plasma pharmacokinetics of a polypeptide, the method comprising changing the isoelectric point of the polypeptide comprising an antibody variable region by the method described in any one of [1] to [8];

[11][10] The method described in claim 1, wherein the pharmacokinetic control refers to an increase or decrease in any one of the parameters of plasma clearance (CL), area under the concentration curve (AUC), mean plasma residence time, and plasma half-life (t1/2);

[12] A polypeptide comprising a variable region of an antibody obtained by the method of [10] and having controlled pharmacokinetics in plasma;

[13] A method for producing a polypeptide comprising an antibody variable region having an altered isoelectric point, the method comprising:

(a) modifying a nucleic acid encoding a polypeptide comprising amino acid residues to change the charge of at least one of the amino acid residues that can be exposed on the surface of the CDR region of the polypeptide;

(b) culturing the host cell to express the nucleic acid;

(c) recovering the polypeptide comprising the antibody variable region from the host cell culture;

[14] [13] The method described, wherein the polypeptide comprising an antibody variable region further comprises an FcRn binding region;

[15] The method described in [13], wherein the polypeptide comprising an antibody variable region is an IgG antibody;

[16] The method of [13], wherein the polypeptide comprising an antibody variable region is a chimeric antibody, a humanized antibody or a human antibody;

[17] The method of [13], wherein the polypeptide comprising an antibody variable region is a multispecific polypeptide that binds to at least two antigens;

[18] The method of [13], wherein the change in the charge of the amino acid residue is an amino acid substitution;

[19] The method of [13], wherein the change in the charge of the amino acid residue is a change that causes the theoretical isoelectric point to change by 1.0 or more;

[20][13] The method described in claim 13, wherein the amino acid residue capable of being exposed on the surface of the CDR region is at least one amino acid residue selected from the group consisting of amino acid residues at positions 31, 61, 62, 64 and 65 according to Kabat numbering in the heavy chain variable region or amino acid residues at positions 24, 27, 53, 54 and 55 according to Kabat numbering in the light chain variable region;

[21] A polypeptide comprising a variable region of an antibody having a modified isoelectric point, obtained by the method of any one of [13] to [20];

[22] A method for producing a polypeptide comprising an antibody variable region whose pharmacokinetics in plasma are controlled, the method comprising: altering the isoelectric point of the polypeptide comprising the antibody variable region by the method described in any one of [13] to [20];

[23][22] The method described in, wherein the pharmacokinetic control refers to an increase or decrease in any one of the parameters of plasma clearance (CL), area under the concentration curve (AUC), mean plasma residence time, and plasma half-life (t1/2);

[24] A polypeptide comprising a variable region of an antibody produced by the method of [22] and having controlled pharmacokinetics in plasma;

[25] A method for producing a multispecific polypeptide, wherein the multispecific polypeptide comprises a first polypeptide having an antibody variable region and a second polypeptide, the method comprising:

(a) modifying a nucleic acid encoding a polypeptide comprising amino acid residues to change the charge of at least one of the amino acid residues that can be exposed on the surface of the CDR region of the polypeptide; the modification of the nucleic acid refers to modifying the nucleic acid encoding the amino acid residues of the first polypeptide and/or the nucleic acid encoding the amino acid residues of the second polypeptide so that the difference in isoelectric point between the first polypeptide and the second polypeptide is increased compared to before the modification;

(b) culturing the host cell to express the nucleic acid;

(c) recovering the multispecific antibody from the host cell culture;

[26] [25], wherein the step of recovering the multispecific polypeptide comprising the first polypeptide and the second polypeptide from the host cell culture is performed according to a standard chromatography method;

[27] The method of [25], wherein, in the modification of the nucleic acid, the nucleic acid is modified so that the peaks obtained in a standard chromatography analysis for the homomultimer of the first polypeptide, the homomultimer of the second polypeptide, and the heteromultimer of the first polypeptide and the second polypeptide become more separated peaks than those before the modification;

[28] The method described in [25], wherein the multispecific polypeptide is a multispecific antibody;

[29] A multispecific antibody produced according to the method described in [27];

[30] The multispecific antibody described in [29], wherein the multispecific antibody is a bispecific antibody;

[31] An antibody comprising a CDR selected from a human-derived CDR, a non-human-derived CDR, and a synthetic CDR, a human-derived framework region (FR), and a human constant region, wherein at least one amino acid residue capable of being exposed on the surface of the CDR has an amino acid residue with a different charge from that of the amino acid residue at the position corresponding to the wild-type CDR, and the isoelectric point of the antibody is altered while maintaining antigen-binding activity compared to the antibody before modification;

[32] The antibody of [31], wherein the human constant region comprises a human Fc region;

[33] [31] The antibody described in which the pharmacokinetics in plasma are controlled by changing the isoelectric point;

[34] an IgG antibody, wherein the charge of at least one amino acid residue selected from amino acid residues 31, 61, 62, 64, and 65 according to Kabat numbering in the heavy chain variable region or amino acid residues 24, 27, 53, 54, and 55 according to Kabat numbering in the light chain variable region of the IgG antibody is altered, and the isoelectric point is altered compared to before modification of the amino acid residue;

[35] [34] The antibody described in which the modified amino acid residue is selected from the group consisting of the amino acid residues in either group (a) or (b):

(a) Glutamic acid (E), aspartic acid (D);

(b) lysine (K), arginine (R), and histidine (H);

[36] A multispecific antibody comprising a first polypeptide and a second polypeptide, wherein at least one amino acid residue selected from amino acid residues 31, 61, 62, 64, and 65 according to Kabat numbering in the heavy chain variable region of the first polypeptide or amino acid residues 24, 27, 53, 54, and 55 according to Kabat numbering in the light chain variable region of the first polypeptide is charged, and the isoelectric points of the first polypeptide and the second polypeptide are different;

[37][36] The antibody described in, wherein at least one amino acid residue selected from the group consisting of amino acid residues at positions 31, 61, 62, 64, and 65 according to Kabat numbering in the heavy chain variable region of the second polypeptide or amino acid residues at positions 24, 27, 53, 54, and 55 according to Kabat numbering in the light chain variable region has a charge opposite to that of the amino acid residue selected in the first polypeptide, or has no charge;

[38] The antibody described in [36], wherein the combination of the charged amino acid residue and the amino acid residue having an opposite charge to the amino acid residue is selected from the amino acid residues contained in any one of the following groups (a) or (b):

(a) Glutamic acid (E), aspartic acid (D);

(b) lysine (K), arginine (R), and histidine (H);

[39] The antibody described in [36] is a multispecific antibody comprising the first polypeptide and the second polypeptide, wherein the homologous multimers of the first polypeptide and the homologous multimers of the second polypeptide form separated peaks in standard chromatographic analysis;

[40] A composition comprising the antibody of any one of [31] to [39] and a pharmaceutically acceptable carrier;

[41] A nucleic acid encoding a polypeptide constituting the antibody described in any one of [31] to [39];

[42] A host cell having the nucleic acid described in [41];

[43] The method for producing an antibody according to any one of [31] to [39], comprising: culturing the host cell according to [42], and recovering the polypeptide from the cell culture;

[44] A method for replacing amino acid residues, which method refers to replacing amino acid residues that can be exposed on the surface of the complementarity determining region (CDR) of a polypeptide comprising an antibody variable region while maintaining the antigen-binding activity of the polypeptide, in which at least one amino acid residue selected from the amino acid residues at positions 31, 61, 62, 64 and 65 according to Kabat numbering in the heavy chain variable region or the amino acid residues at positions 24, 27, 53, 54 and 55 according to Kabat numbering in the light chain variable region is substituted.

The present invention also provides the following [1] to [38].

[1] A method for producing a glypican 3 antibody with controlled hemodynamics, the method comprising the following steps:

(a) modifying a nucleic acid encoding at least one amino acid residue to change the charge of at least one amino acid residue that can be exposed on the surface of a glypican 3 antibody;

(b) culturing the host cell containing the nucleic acid to express the nucleic acid;

(c) recovering the Glypican 3 antibody from the culture of the host cell;

[2] The method of [1], wherein the control of blood dynamics refers to an increase or decrease in any one of the parameters of blood half-life, mean blood residence time, and blood clearance rate;

[3] The method of [1], wherein the change in the charge of the amino acid residue in step (a) is performed by amino acid substitution;

[4] The method of [1], wherein the amino acid residue capable of being exposed on the surface of the Glypican 3 antibody is located in a region other than the FcRn-binding region of the Glypican 3 antibody;

[5] [4], wherein the FcRn binding region comprises an Fc region;

[6] [1], wherein the anti-glypican 3 antibody is an IgG antibody;

[7][6], wherein the amino acid residue whose charge is altered is an amino acid residue in the heavy chain variable region or the light chain variable region of an IgG antibody;

[8][7] The method described in claim 8 is characterized in that: the above-mentioned Glypican 3 antibody is a Glypican 3 antibody comprising a complementarity determining region (CDR), a human-derived framework region (FR), and a human constant region; the change in the charge of the amino acid residue in step (a) refers to modifying at least one amino acid residue in the CDR or FR of the antibody to be modified that can be exposed on the antibody surface into an amino acid residue with a different charge from that of the amino acid residue;

[9] The method of [8], wherein the above-mentioned glypican 3 antibody is an antibody with reduced fucose content bound to its Fc region;

[10] A glypican 3 antibody produced by the method described in any one of [1] to [9];

[11] A method for stabilizing a glypican 3 antibody, characterized in that at least one amino acid residue constituting the glypican 3 antibody comprising a complementarity determining region (CDR), a human-derived framework region (FR), and a human constant region is modified to increase the Tm value of the antibody; the stabilization method comprises:

(a) modifying a nucleic acid encoding at least one amino acid residue to increase the Tm value of a modified Glypican 3 antibody;

(b) culturing the host cell containing the nucleic acid to express the nucleic acid;

(c) recovering the antibody from the culture of the host cell;

[12][11] The method according to claim 1, wherein the amino acid residue in step (a) is present in the FR1 region and/or FR2 region of the H chain or L chain;

[13] The method of [12], characterized in that: the amino acid residues in the FR2 region of the H chain described in [12] are replaced with amino acid residues in the FR2 region of the VH4 subclass;

[14] The method described in [12], characterized in that: the amino acid residues in the L chain FR2 region described in [12] are replaced with amino acid residues in the FR2 region of the VK3 subclass;

[15] A method for controlling the cytotoxicity of an antibody, the method comprising the following steps:

(a) modifying a nucleic acid encoding at least one amino acid residue to change the charge of at least one amino acid residue that can be exposed on the surface of a cytotoxic antibody;

(b) culturing the host cell containing the nucleic acid to express the nucleic acid;

(c) recovering the antibody from the culture of the host cell;

[16][15] The method described in, wherein the above-mentioned control of blood dynamics refers to the control of any one parameter of blood half-life, mean blood residence time, and blood clearance rate;

[17] The method of [15], wherein the charge of the amino acid residue in step (a) is changed by amino acid substitution;

[18] The method of [15], wherein the amino acid residue capable of being exposed on the antibody surface is located in a region other than the FcRn-binding region of the antibody;

[19] [18], wherein the FcRn binding region comprises an Fc region;

[20][15] The method described, wherein the glypican 3 antibody is an IgG antibody;

[21][20] The method described, wherein the amino acid residue whose charge is changed is an amino acid residue in the heavy chain variable region or the light chain variable region of an IgG antibody;

[22][21] The method is characterized in that: the above-mentioned antibody is an antibody comprising a complementarity determining region (CDR) derived from an animal other than human, a framework region (FR) derived from human, and a human constant region; the change in the charge of the amino acid residue in step (a) refers to modifying at least one amino acid residue in the CDR or FR of the antibody to be modified that can be exposed on the antibody surface to an amino acid residue having a different charge from that amino acid residue;

[23] The method of [22], wherein the antibody is an antibody with reduced fucose content bound to its Fc region;

[24] An antibody produced by the method of any one of [15] to [23];

[25] [24] The antibody, wherein the antibody is a glypican 3 antibody;

[26] An antibody comprising an H chain V region and an L chain V region, wherein the amino acid residues constituting the H chain V region shown in SEQ ID NO: 195 are substituted with any one or more of the following:

(a) replacing amino acid residue K at position 19 with T;

(b) substituting amino acid residue Q at position 43 into E;

(c) substituting amino acid residue K at position 63 into S;

(d) substituting amino acid residue K at position 65 into Q;

(e) substituting amino acid residue G at position 66 into D;

In the L chain V region, any one or more of the following substitutions are made to the amino acid residues constituting the L chain V region shown in SEQ ID NO: 201:

(f) substitution of amino acid residue Q at position 27 with E;

(g) substitution of amino acid residue K at position 79 with T;

(h) substitution of amino acid residue R at position 82 with S;

[27] The antibody of [26], comprising the H chain represented by SEQ ID NO: 197 and the L chain represented by SEQ ID NO: 203;

[28] The antibody of [26], comprising the H chain represented by SEQ ID NO: 198 and the L chain represented by SEQ ID NO: 204;

[29] An antibody comprising an H chain V region and an L chain V region, wherein the amino acid residues constituting the H chain V region shown in SEQ ID NO: 195 are substituted with any one or more of the following:

(a) replacing amino acid residue Q at position 43 with K;

(b) replacing amino acid residue D at position 52 with N;

(c) substitution of amino acid residue Q at position 107 with R;

In the L chain V region, any one or more of the following substitutions are made to the amino acid residues constituting the L chain V region shown in SEQ ID NO: 201:

(d) substituting amino acid residue E at position 17 into Q;

(e) substitution of amino acid residue Q at position 27 with R;

(f) substitution of amino acid residue Q at position 105 with R;

[30] The antibody of [29], comprising the H chain variable region represented by SEQ ID NO: 198 and the L chain variable region represented by SEQ ID NO: 204;

[31] The antibody of [29], comprising the H chain variable region represented by SEQ ID NO: 199 and the L chain variable region represented by SEQ ID NO: 205;

[32] The antibody according to any one of [26] to [31], comprising a C region of a human antibody;

[33] A composition comprising the antibody of [32] and a pharmaceutically acceptable carrier;

[34] A cancer therapeutic drug comprising the antibody described in [32] as an active ingredient;

[35] [34] The cancer therapeutic drug, wherein the cancer is liver cancer;

[36] A nucleic acid encoding a polypeptide constituting the antibody described in any one of [26] to [31];

[37] A host cell containing the nucleic acid described in [36];

[38] A method for producing an antibody according to any one of [26] to [31], comprising: culturing the host cell according to [37]; and recovering the polypeptide from the cell culture.

The present invention also provides the following [1] to [41].

[1] The anti-IL-6 receptor antibody according to any one of (a) to (y) below:

(a) an antibody comprising a heavy chain variable region having a CDR1, wherein Ser at position 1 in the amino acid sequence of SEQ ID NO: 1 is substituted with another amino acid;

(b) an antibody comprising a heavy chain variable region having a CDR1, wherein the Trp at position 5 in the amino acid sequence of SEQ ID NO: 1 is substituted with another amino acid;

(c) an antibody comprising a heavy chain variable region having a CDR2, wherein Tyr at position 1 in the amino acid sequence of SEQ ID NO: 2 is substituted with another amino acid;

(d) an antibody comprising a heavy chain variable region having a CDR2, wherein Thr at position 8 in the amino acid sequence of SEQ ID NO: 2 is substituted with another amino acid;

(e) an antibody comprising a heavy chain variable region having a CDR2, wherein Thr at position 9 in the amino acid sequence of SEQ ID NO: 2 is substituted with another amino acid;

(f) an antibody comprising a heavy chain variable region having a CDR3, wherein Ser at position 1 in the amino acid sequence of SEQ ID NO: 3 is substituted with another amino acid;

(g) an antibody comprising a heavy chain variable region having a CDR3, wherein Leu at position 2 in the amino acid sequence of SEQ ID NO: 3 is substituted with another amino acid;

(h) an antibody comprising a heavy chain variable region having a CDR3, wherein Thr at position 5 in the amino acid sequence of SEQ ID NO: 3 is substituted with another amino acid;

(i) an antibody comprising a heavy chain variable region having a CDR3, wherein Ala at position 7 in the amino acid sequence of SEQ ID NO: 3 is substituted with another amino acid;

(j) an antibody comprising a heavy chain variable region having a CDR3, wherein Met at position 8 in the amino acid sequence of SEQ ID NO: 3 is substituted with another amino acid;

(k) an antibody comprising a heavy chain variable region having a CDR3, wherein Ser at position 1 and Thr at position 5 in the amino acid sequence of SEQ ID NO: 3 are substituted with other amino acids;

(1) an antibody comprising a heavy chain variable region having a CDR3, wherein Leu at position 2, Ala at position 7, and Met at position 8 in the amino acid sequence of SEQ ID NO: 3 are substituted with other amino acids;

(m) an antibody comprising a light chain variable region having a CDR1, wherein the Arg at position 1 in the amino acid sequence of SEQ ID NO: 4 is substituted with another amino acid;

(n) an antibody comprising a light chain variable region having a CDR1, wherein the Gln at position 4 in the amino acid sequence of SEQ ID NO: 4 is substituted with another amino acid;

(o) an antibody comprising a light chain variable region having a CDR1, wherein Tyr at position 9 in the amino acid sequence of SEQ ID NO: 4 is substituted with another amino acid;

(p) an antibody comprising a light chain variable region having a CDR1, wherein Asn at position 11 in the amino acid sequence of SEQ ID NO: 4 is substituted with another amino acid;

(q) an antibody comprising a light chain variable region having a CDR2, wherein Thr at position 2 in the amino acid sequence of SEQ ID NO: 5 is substituted with another amino acid;

(r) an antibody comprising a light chain variable region having a CDR3, wherein the Gln at position 1 in the amino acid sequence of SEQ ID NO: 6 is substituted with another amino acid;

(s) an antibody comprising a light chain variable region having a CDR3, wherein Gly at position 3 in the amino acid sequence of SEQ ID NO: 6 is substituted with another amino acid;

(t) an antibody comprising a light chain variable region comprising CDR1 and CDR3, wherein Tyr at position 9 in the amino acid sequence of SEQ ID NO: 4 is substituted with another amino acid, and Gly at position 3 in the amino acid sequence of SEQ ID NO: 6 is substituted with another amino acid;

(u) an antibody comprising a light chain variable region having a CDR3, wherein Thr at position 5 in the amino acid sequence of SEQ ID NO: 6 is substituted with another amino acid;

(v) an antibody comprising a light chain variable region having a CDR3, wherein Gln at position 1 and Thr at position 5 in the amino acid sequence of SEQ ID NO: 6 are substituted with other amino acids;

(w) an antibody comprising a heavy chain variable region comprising CDR2 and CDR3, wherein Thr at position 9 in the amino acid sequence of SEQ ID NO: 2 is substituted with another amino acid, and Ser at position 1 and Thr at position 5 in the amino acid sequence of SEQ ID NO: 3 are substituted with other amino acids;

(x) an antibody comprising the heavy chain variable region described in (k) and the light chain variable region described in (v); or

(y) The antibody described in (x), further comprising the CDR2 described in (e).

[2] An anti-IL-6 receptor antibody comprising a light chain variable region having a CDR2, wherein Thr at position 2 in the amino acid sequence of SEQ ID NO: 5 is substituted with another amino acid.

[3] The anti-IL-6 receptor antibody according to any one of (a) to (y) below:

(a) an antibody comprising a heavy chain variable region having FR1, wherein Arg at position 13 in the amino acid sequence of SEQ ID NO: 7 is substituted with another amino acid;

(b) an antibody comprising a heavy chain variable region having FR1, wherein Gln at position 16 in the amino acid sequence of SEQ ID NO: 7 is substituted with another amino acid;

(c) an antibody comprising a heavy chain variable region having FR1, wherein Thr at position 23 in the amino acid sequence of SEQ ID NO: 7 is substituted with another amino acid;

(d) an antibody comprising a heavy chain variable region having FR1, wherein Thr at position 30 in the amino acid sequence of SEQ ID NO: 7 is substituted with another amino acid;

(e) an antibody comprising a heavy chain variable region having FR1, wherein Arg at position 13, Gln at position 16, Thr at position 23, and Thr at position 30 in the amino acid sequence of SEQ ID NO: 7 are substituted with other amino acids;

(f) an antibody comprising a heavy chain variable region having FR2, wherein Arg at position 8 in the amino acid sequence of SEQ ID NO: 8 is substituted with another amino acid;

(g) an antibody comprising a heavy chain variable region having FR3, wherein Met at position 4 in the amino acid sequence of SEQ ID NO: 9 is substituted with another amino acid;

(h) an antibody comprising a heavy chain variable region having FR3, wherein Leu at position 5 in the amino acid sequence of SEQ ID NO: 9 is substituted with another amino acid;

(i) an antibody comprising a heavy chain variable region having FR3, wherein Arg at position 16 in the amino acid sequence of SEQ ID NO: 9 is substituted with another amino acid;

(j) an antibody comprising a heavy chain variable region having FR3, wherein Val at position 27 in the amino acid sequence of SEQ ID NO: 9 is substituted with another amino acid;

(k) an antibody comprising a heavy chain variable region having FR3, wherein Met at position 4, Leu at position 5, Arg at position 16, and Val at position 27 in the amino acid sequence of SEQ ID NO: 9 are substituted with other amino acids;

(1) an antibody comprising a heavy chain variable region having FR4, wherein Gln at position 3 in the amino acid sequence of SEQ ID NO: 10 is substituted with another amino acid;

(m) an antibody comprising a light chain variable region having FR1, wherein Arg at position 18 in the amino acid sequence of SEQ ID NO: 11 is substituted with another amino acid;

(n) an antibody comprising a light chain variable region having FR2, wherein Lys at position 11 in the amino acid sequence of SEQ ID NO: 12 is substituted with another amino acid;

(o) an antibody comprising a light chain variable region having FR3, wherein Gln at position 23 in the amino acid sequence of SEQ ID NO: 13 is substituted with another amino acid;

(p) an antibody comprising a light chain variable region having FR3, wherein the Pro at position 24 in the amino acid sequence of SEQ ID NO: 13 is substituted with another amino acid;

(q) an antibody comprising a light chain variable region having FR3, wherein Ile at position 27 in the amino acid sequence of SEQ ID NO: 13 is substituted with another amino acid;

(r) an antibody comprising a light chain variable region having FR3, wherein Gln at position 23, Pro at position 24, and Ile at position 27 in the amino acid sequence of SEQ ID NO: 13 are substituted with other amino acids;

(s) an antibody comprising a light chain variable region having FR4, wherein Lys at position 10 in the amino acid sequence of SEQ ID NO: 14 is substituted with another amino acid;

(t) an antibody comprising a heavy chain variable region having FR4, wherein Ser at position 5 in the amino acid sequence of SEQ ID NO: 10 is substituted with another amino acid;

(u) an antibody comprising a heavy chain variable region having FR4, wherein Gln at position 3 and Ser at position 5 in the amino acid sequence of SEQ ID NO: 10 are substituted with other amino acids;

(v) an antibody comprising a heavy chain variable region having FR3, wherein the FR3 has the amino acid sequence set forth in SEQ ID NO: 184;

(w) an antibody comprising a heavy chain variable region comprising FR1 of (e), FR2 of (f), FR3 of (k), and FR4 of (l) or (u);

(x) an antibody comprising a light chain variable region comprising FR1 of (m), FR2 of (n), FR3 of (r), and FR4 of (s); or

(y) An antibody comprising the heavy chain variable region described in (w) and the light chain variable region described in (x).

[4] The anti-IL-6 receptor antibody according to any one of (a) to (l) below:

(a) an antibody comprising a heavy chain variable region having a CDR1, wherein Ser at position 1 in the amino acid sequence of SEQ ID NO: 1 is substituted with another amino acid;

(b) an antibody comprising a heavy chain variable region having a CDR2, wherein Thr at position 9 in the amino acid sequence of SEQ ID NO: 2 is substituted with another amino acid;

(c) an antibody comprising a heavy chain variable region having a CDR2, wherein Ser at position 16 in the amino acid sequence of SEQ ID NO: 2 is substituted with another amino acid;

(d) an antibody comprising a heavy chain variable region having a CDR2, wherein Thr at position 9 and Ser at position 16 in the amino acid sequence of SEQ ID NO: 2 are substituted with other amino acids;

(e) an antibody comprising a light chain variable region having a CDR1, wherein the Arg at position 1 in the amino acid sequence of SEQ ID NO: 4 is substituted with another amino acid;

(f) an antibody comprising a light chain variable region having a CDR2, wherein Thr at position 2 in the amino acid sequence of SEQ ID NO: 5 is substituted with another amino acid;

(g) an antibody comprising a light chain variable region having a CDR2, wherein Arg at position 4 in the amino acid sequence of SEQ ID NO: 5 is substituted with another amino acid;

(h) an antibody comprising a light chain variable region having a CDR2, wherein Thr at position 2 and Arg at position 4 in the amino acid sequence of SEQ ID NO: 5 are substituted with other amino acids;

(i) an antibody comprising a light chain variable region having a CDR3, wherein Thr at position 5 in the amino acid sequence of SEQ ID NO: 6 is substituted with another amino acid;

(j) an antibody comprising a heavy chain variable region comprising the CDR1 described in (a), the CDR2 described in (d), and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 3;

(k) an antibody comprising a light chain variable region comprising the CDR1 of (e), the CDR2 of (h), and the CDR3 of (i); or

(l) An antibody comprising the heavy chain variable region described in (j) and the light chain variable region described in (k).

[5] The anti-IL-6 receptor antibody according to any one of (a) to (f) below:

(a) an antibody comprising a heavy chain variable region comprising CDR1, CDR2, and CDR3, wherein Ser at position 1 of the CDR1 in the amino acid sequence set forth in SEQ ID NO: 1 is substituted with another amino acid, Thr at position 9 and Ser at position 16 of the CDR2 in the amino acid sequence set forth in SEQ ID NO: 2 are substituted with other amino acids, and Ser at position 1 and Thr at position 5 of the CDR3 in the amino acid sequence set forth in SEQ ID NO: 3 are substituted with other amino acids;

(b) an antibody comprising a light chain variable region comprising CDR1, CDR2, and CDR3, wherein Arg at position 1 of the CDR1 in the amino acid sequence set forth in SEQ ID NO: 4 is substituted with another amino acid, Thr at position 2 and Arg at position 4 of the CDR2 in the amino acid sequence set forth in SEQ ID NO: 5 are substituted with other amino acids, and Gln at position 1 and Thr at position 5 of the CDR3 in the amino acid sequence set forth in SEQ ID NO: 6 are substituted with other amino acids;

(c) an antibody comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 22;

(d) an antibody comprising a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 23;

(e) an antibody comprising the heavy chain variable region described in (a) and the light chain variable region described in (b); or

(f) An antibody comprising the heavy chain variable region described in (c) and the light chain variable region described in (d).

[6] The human antibody constant region according to any one of (a) to (c) below:

(a) A human antibody constant region characterized in that: in the amino acid sequence of SEQ ID NO: 19, both the Gly at position 329 (EU numbering position 446) and the Lys at position 330 (EU numbering position 447) are deleted;

(b) A human antibody constant region characterized in that: in the amino acid sequence of SEQ ID NO: 20, both the Gly at position 325 (EU numbering position 446) and the Lys at position 326 (EU numbering position 447) are deleted;

(c) A human antibody constant region characterized in that: in the amino acid sequence described in SEQ ID NO: 21, both Gly at position 326 (EU numbering position 446) and Lys at position 327 (EU numbering position 447) are deleted.

[7] An IgG2 constant region, wherein the amino acids at positions 209 (EU numbering position 330), 210 (EU numbering position 331), and 218 (EU numbering position 339) in the amino acid sequence of SEQ ID NO: 20 are substituted with other amino acids.

[8] An IgG2 constant region, wherein the amino acid at position 276 (EU numbering position 397) in the amino acid sequence of SEQ ID NO: 20 is substituted with another amino acid.

[9] An IgG2 constant region, wherein the amino acid at position 14 (EU numbering position 131), the amino acid at position 102 (EU numbering position 219) and/or the amino acid at position 16 (EU numbering position 133) in the amino acid sequence of SEQ ID NO: 20 is substituted with another amino acid.

[10] The IgG2 constant region described in [9] is characterized in that: in the amino acid sequence recorded in SEQ ID NO: 20, the amino acids at positions 20 (EU numbering position 137) and 21 (EU numbering position 138) are further substituted with other amino acids.

[11] An IgG2 constant region, wherein in the amino acid sequence of SEQ ID NO: 20, His at position 147 (EU numbering position 268), Arg at position 234 (EU numbering position 355), and/or Gln at position 298 (EU numbering position 419) are substituted with other amino acids;

[12] An IgG2 constant region having the following amino acid sequence: in the amino acid sequence described in SEQ ID NO: 20, the amino acids at positions 209 (EU numbering position 330), 210 (EU numbering position 331), 218 (EU numbering position 339), 276 (EU numbering position 397), 14 (EU numbering position 131), 16 (EU numbering position 133), 102 (EU numbering position 219), 20 (EU numbering position 137), and 21 (EU numbering position 138) are substituted with other amino acids.

[13] An IgG2 constant region having the following amino acid sequence: the IgG2 constant region described in [12], further comprising an amino acid sequence in which both Gly at position 325 (EU numbering position 446) and Lys at position 326 (EU numbering position 447) are deleted.

[14] An IgG2 constant region having the following amino acid sequence: in the amino acid sequence described in SEQ ID NO: 20, the amino acids at positions 276 (EU numbering position 397), 14 (EU numbering position 131), 16 (EU numbering position 133), 102 (EU numbering position 219), 20 (EU numbering position 137) and 21 (EU numbering position 138) are substituted with other amino acids.

[15] An IgG2 constant region having the following amino acid sequence: the IgG2 constant region described in [14], further comprising an amino acid sequence in which both Gly at position 325 (EU numbering position 446) and Lys at position 326 (EU numbering position 447) are deleted.

[16] An IgG2 constant region having the following amino acid sequence: in the amino acid sequence described in SEQ ID NO: 20, Cys at position 14 (EU numbering position 131), Arg at position 16 (EU numbering position 133), Cys at position 102 (EU numbering position 219), Glu at position 20 (EU numbering position 137), Ser at position 21 (EU numbering position 138), His at position 147 (EU numbering position 268), Arg at position 234 (EU numbering position 355), and Gln at position 298 (EU numbering position 419) are substituted with other amino acids.

[17] An IgG2 constant region having the following amino acid sequence: the IgG2 constant region described in [16], further comprising an amino acid sequence in which both Gly at position 325 (EU numbering position 446) and Lys at position 326 (EU numbering position 447) are deleted.

[18] An IgG2 constant region having the following amino acid sequence: in the amino acid sequence described in SEQ ID NO: 20, Cys at position 14 (EU numbering position 131), Arg at position 16 (EU numbering position 133), Cys at position 102 (EU numbering position 219), Glu at position 20 (EU numbering position 137), Ser at position 21 (EU numbering position 138), His at position 147 (EU numbering position 268), Arg at position 234 (EU numbering position 355), Gln at position 298 (EU numbering position 419), and Asn at position 313 (EU numbering position 434) are substituted with other amino acids.

[19] An IgG2 constant region having the following amino acid sequence: the IgG2 constant region described in [18], further comprising an amino acid sequence in which both Gly at position 325 (EU numbering position 446) and Lys at position 326 (EU numbering position 447) are deleted.

[20] An IgG4 constant region, characterized in that the amino acid at position 289 (EU numbering position 409) in the amino acid sequence of SEQ ID NO: 21 is substituted with another amino acid.

[21] An IgG4 constant region having the following amino acid sequence: an amino acid sequence in which, in the amino acid sequence set forth in SEQ ID NO: 21, amino acids at position 289 (EU numbering position 409), amino acids 14, 16, 20, 21, 97, 100, 102, 103, 104, and 105 (EU numbering positions 131, 133, 137, 138, 214, 217, 219, 220, 221, and 222, respectively), and amino acids 113, 114, and 115 (EU numbering positions 233, 234, and 235, respectively) are substituted with other amino acids, and the amino acid at position 116 (EU numbering position 236) is deleted.

[22] An IgG4 constant region, wherein the Gly at position 326 (EU numbering position 446) and the Lys at position 327 (EU numbering position 447) are further deleted in the IgG4 constant region described in [21].

[23] An IgG2 constant region having the following amino acid sequence: in the amino acid sequence described in SEQ ID NO: 20, Ala at position 209 (EU numbering position 330), Pro at position 210 (EU numbering position 331), Thr at position 218 (EU numbering position 339), Cys at position 14 (EU numbering position 131), Arg at position 16 (EU numbering position 133), Cys at position 102 (EU numbering position 219), Glu at position 20 (EU numbering position 137), and Ser at position 21 (EU numbering position 138) are substituted with other amino acids.

[24] An IgG2 constant region having the following amino acid sequence: the IgG2 constant region described in [23], further comprising an amino acid sequence in which both Gly at position 325 (EU numbering position 446) and Lys at position 326 (EU numbering position 447) are deleted.

[25] An IgG2 constant region having the following amino acid sequence: in the amino acid sequence described in SEQ ID NO: 20, Cys at position 14 (EU numbering position 131), Arg at position 16 (EU numbering position 133), Cys at position 102 (EU numbering position 219), Glu at position 20 (EU numbering position 137), and Ser at position 21 (EU numbering position 138) are substituted with other amino acids.

[26] An IgG2 constant region having the following amino acid sequence: the IgG2 constant region described in [25], further comprising an amino acid sequence in which both Gly at position 325 (EU numbering position 446) and Lys at position 326 (EU numbering position 447) are deleted.

[27] A constant region having the amino acid sequence set forth in SEQ ID NO: 24.

[28] A constant region having the amino acid sequence set forth in SEQ ID NO: 118.

[29] A constant region having the amino acid sequence set forth in SEQ ID NO: 25.

[30] A constant region having the amino acid sequence set forth in SEQ ID NO: 151.

[31] A constant region having the amino acid sequence set forth in SEQ ID NO: 152.

[32] A constant region having the amino acid sequence set forth in SEQ ID NO: 153.

[33] A constant region having the amino acid sequence set forth in SEQ ID NO: 164.

[34] A human antibody constant region having the amino acid sequence set forth in SEQ ID NO: 194 (M40ΔGK).

[35] A human antibody constant region having the amino acid sequence set forth in SEQ ID NO: 192 (M86ΔGK).

[36] An antibody comprising the constant region according to any one of [6] to [35].

The antibody described in [37][36] is characterized in that it binds to the IL-6 receptor.

[38] An anti-human IL-6 receptor antibody, wherein the antibody has an IL-6 receptor binding activity of 1 nM or less.

[39] An anti-human IL-6 receptor antibody, wherein the measured isoelectric point of the full-length antibody is 7.0 or less, or the theoretical isoelectric point of the variable region is 5.0 or less.

[40] An anti-IL-6 receptor antibody, characterized in that: in a buffer solution of 20 mM histidine-HCl, 150 mM NaCl, pH 6.5-7.0, at an antibody concentration of 100 mg/mL, at 25°C for one month, the increase in the aggregation ratio of the antibody is less than 0.3%.

[41] A pharmaceutical composition comprising the antibody according to any one of [36] to [40].

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a graph showing the BaF/gp130 neutralization activity of WT and RD_6.

FIG2 is a graph showing a sensorgram of the interaction between rhIL-s6R (R&D Systems) and WT.

FIG3 is a graph showing the sensorgram of the interaction of rhIL-s6R (R&D Systems) with RD_6.

FIG4-1 is a diagram showing a list of CDR mutations that improve affinity and neutralization activity compared to WT.

Figure 4-2 is a continuation of Figure 4-1.

FIG5 is a diagram showing a list of CDR mutations whose affinity and neutralizing activity are improved by combination.

FIG6 is a graph showing the BaF/gp130 neutralization activities of WT and RDC23.

FIG. 7 is a graph showing a sensorgram of the interaction of rhIL-s6R (R&D Systems) with RDC23.

FIG8 is a graph showing a sensorgram of the interaction of rhsIL-6R with WT.

FIG. 9 is a graph showing a sensorgram of the interaction of rhsIL-6R with RDC23.

FIG. 10 is a graph showing a sensorgram of the interaction of SR344 with WT.

FIG. 11 is a graph showing a sensorgram of the interaction of SR344 and RDC23.

FIG. 12 is a graph showing the BaF/gp130 neutralization activities of WT and H53L28.

FIG. 13 is a graph showing a sensorgram of the interaction of SR344 with H53/L28.

FIG. 14 is a graph showing changes in plasma concentrations of WT and H53/L28 after intravenous administration to mice.

FIG. 15 is a graph showing changes in plasma concentrations of WT and H53/L28 after subcutaneous administration to mice.

FIG. 16 is a graph showing the BaF/gp130 neutralization activities of WT and PF1.

FIG. 17 is a graph showing a sensorgram of the interaction of SR344 and PF1.

FIG18 is a graph showing the results of a high-concentration stability test of WT and PF1.

FIG19 is a graph showing changes in plasma concentrations of WT and PF1 after intravenous administration to human IL-6 receptor transgenic mice.

FIG20 is a graph showing changes in the concentration of unbound human soluble IL-6 receptor after intravenous administration of WT and PF1 to human IL-6 receptor transgenic mice.

FIG21 is a graph showing the results of gel filtration chromatography analysis of the aggregate content of WT-IgG1, WT-IgG2, WT-IgG4, IgG2-M397V, and IgG4-R409K purified by the hydrochloric acid elution method.

FIG22 is a graph showing the results of cation exchange chromatography (IEC) analysis of WT-IgG1, WT-IgG2, and WT-IgG4.

FIG23 is a diagram showing the putative disulfide bond conformation (binding pattern) of the hinge region of WT-IgG2.

FIG24 is a diagram showing the putative disulfide bond conformation of the hinge region of WT-IgG2-SKSC.

FIG25 is a graph showing the results of cation exchange chromatography (IEC) analysis of WT-IgG2 and IgG2-SKSC.

FIG26 is a graph showing the results of cation exchange chromatography (IEC) analysis of humanized PM-1 antibody, H chain C-terminal ΔK antibody, and H chain C-terminal ΔGK antibody.

FIG27 is a graph showing a comparison of the binding amounts of WT-IgG1, WT-IgG2, WT-IgG4, WT-M14ΔGK, WT-M17ΔGK, and WT-M11ΔGK to FcγRI.

FIG28 is a graph showing a comparison of the binding amounts of WT-IgG1, WT-IgG2, WT-IgG4, WT-M14ΔGK, WT-M17ΔGK, and WT-M11ΔGK to FcγRIIa.

FIG29 is a graph showing a comparison of the binding amounts of WT-IgG1, WT-IgG2, WT-IgG4, WT-M14ΔGK, WT-M17ΔGK, and WT-M11ΔGK to FcγRIIb.

FIG30 is a graph showing a comparison of the binding amounts of WT-IgG1, WT-IgG2, WT-IgG4, WT-M14ΔGK, WT-M17ΔGK, and WT-M11ΔGK to FcγRIIIa(Val).

FIG31 is a graph showing the increase in the amount of aggregates in a high-concentration stability test of WT-IgG1, WT-M14ΔGK, WT-M17ΔGK, and WT-M11ΔGK.

FIG32 is a graph showing the increased amount of Fab fragments in a high-concentration stability test of WT-IgG1, WT-M14ΔGK, WT-M17ΔGK, and WT-M11ΔGK.

FIG33 is a graph showing the results of cation exchange chromatography (IEC) analysis of WT-IgG2, WT-M14ΔGK, and WT-M31ΔGK.

FIG34 is a graph showing the BaF/gp130 neutralization activities of WT and F2H/L39-IgG1.

FIG35 is a graph showing changes in plasma antibody concentrations following subcutaneous administration of WT, PF1, and F2H/L39-IgG1 at 1.0 mg/kg to cynomolgus monkeys.

FIG36 is a graph showing changes in CRP concentrations in the WT and F2H/L39-IgG1-administered groups of cynomolgus monkeys.

FIG37 is a graph showing changes in the concentration of non-binding cynomolgus monkey IL-6 receptor in the WT and F2H/L39-IgG1-administered groups of cynomolgus monkeys.

Fig. 38 is a graph showing changes in plasma concentrations of WT-IgG1 and WT-M14 following intravenous administration to human FcRn transgenic mice.

FIG39 is a graph showing changes in plasma concentrations of WT-IgG1, WT-M14, and WT-M58 after intravenous administration to human FcRn transgenic mice.

FIG40 is a graph showing changes in plasma concentrations of WT-IgG1, WT-M44, WT-M58, and WT-M73 after intravenous administration to human FcRn transgenic mice.

FIG41 is a graph showing the effect of the constant regions of anti-IL-6 receptor antibody WT, anti-IL-6 receptor antibody F2H/L39, anti-IL-31 receptor antibody H0L0, and anti-RANKL antibody DNS on heterogeneity evaluated by cation exchange chromatography.

FIG42 is a graph showing the effect of cysteine residues in the CH1 domain of anti-IL-6 receptor antibody WT and anti-IL-6 receptor antibody F2H/L39 on heterogeneity evaluated by cation exchange chromatography.

FIG43 is a graph showing the effect of cysteine in the CH1 domain of anti-IL-6 receptor antibody WT on the denaturation peak evaluated by DSC.

FIG44 is a graph showing the neutralization activities of TOCILIZUMAB, a control, and Fv5-M83 against BaF/g130.

FIG45 is a graph showing the neutralizing activities of TOCILIZUMAB, Fv3-M73, and Fv4-M73 against BaF/gp130.

FIG46 is a graph showing changes in plasma concentrations of TOCILIZUMAB, a control, Fv3-M73, Fv4-M73, and Fv5-M83 after intravenous administration to cynomolgus monkeys.

FIG47 is a graph showing changes in CRP concentration after intravenous administration of TOCILIZUMAB, a control, Fv3-M73, Fv4-M73, and Fv5-M83 to cynomolgus monkeys.

Fig. 48 is a graph showing changes in the neutralization rate of soluble IL-6 receptor after intravenous administration of TOCILIZUMAB, a control, Fv3-M73, Fv4-M73, and Fv5-M83 to cynomolgus monkeys.

FIG49 is a graph obtained by DSC (differential scanning calorimetry) measurement of the Hspu2.2Lspu2.2 (Hu2.2Lu2.2) antibody.

FIG50 is an electrophoretic diagram of the H0L0 antibody and the Hspu2.2Lspu2.2 (Hu2.2Lu2.2) antibody in high pI isoelectric point electrophoresis.

FIG51 is an electrophoretic diagram of the H0L0 antibody and the Hspd1.8Lspd1.6 (Hd1.8Ld1.6) antibody in low pI isoelectric point electrophoresis.

FIG52 is a graph showing the binding activities of the H15L4 antibody and the H0L0 antibody to glypican 3 (antigen) measured by competition ELISA.

FIG53 is a graph showing the binding activities of Hspu2.2Lspu2.2 (Hu2.2Lu2.2) antibody and H0L0 antibody to glypican 3 (antigen) measured by competition ELISA.

FIG54 is a graph showing the binding activities of Hspd1.8Lspd1.6 (Hd1.8Ld1.6) antibody and H0L0 antibody to glypican 3 (antigen) measured by competition ELISA.

FIG55 shows the anti-tumor effects of the H0L0 antibody, the Hspu2.2Lspu2.2 (Hu2.2Lu2.2) antibody, and the Hspd1.8Lspd1.6 (Hd1.8Ld1.6) antibody in a human liver cancer transplanted mouse model.

FIG56 shows the plasma antibody concentrations of the H0L0 antibody, the Hspu2.2Lspu2.2 (Hu2.2Lu2.2) antibody, and the Hspd1.8Lspd1.6 (Hd1.8Ld1.6) antibody in a human liver cancer transplantation mouse model.

FIG57 shows the ADCC activity of each tested antibody against the human liver cancer cell line Hep G2 cells.

FIG58 is a graph showing the IL-6 receptor neutralizing activity of 6R_b_H1L1, 6R_b_H2L2, 6R_b_H2L3, and 6R_b_H2L4 in BaF/6R.

FIG59 is a graph showing the binding activities of the GPC_H1L1 antibody and the GPC_H2L2 antibody to Glypican 3 (antigen) measured by competition ELISA.

FIG60 is a graph showing the binding activities of the GPC_H2L2 antibody and the GPC_H3L3 antibody to Glypican 3 (antigen) measured by competition ELISA.

Figure 61 is a graph showing the peak separation of A chain-B chain heteromultimers, A chain homomultimers, and B chain homomultimers of 6R_a_H1H3L3, GPC3_H2H3L3, and 31R_H1aH2aL2 measured by cation exchange chromatography.

DETAILED DESCRIPTION

The present invention provides a method for altering the isoelectric point of a polypeptide comprising an antibody variable region while maintaining the antigen-binding activity of the variable region, the method comprising altering the charge of at least one amino acid residue capable of being exposed on the surface of a complementarity-determining region (CDR) of the polypeptide. The present invention also provides a polypeptide comprising an antibody variable region with an altered isoelectric point obtained by this method (e.g., an antibody comprising a CDR selected from a group consisting of a CDR derived from a human, a CDR derived from an animal other than a human, and a synthetic CDR, a framework region (FR) derived from a human, and a human constant region, wherein at least one amino acid residue capable of being exposed on the surface of the CDR is an amino acid residue having a different charge than an amino acid residue at a position corresponding to a wild-type CDR, and the isoelectric point of the antibody is altered while maintaining antigen-binding activity compared to the antibody before modification).

A preferred embodiment of the method of the present invention comprises modifying the charge of at least one amino acid residue that can be exposed on the antibody surface. Specifically, by modifying the charge of the antibody's amino acid residues, the antibody's isoelectric point (pI) is altered, thereby controlling the antibody's plasma pharmacokinetics (blood dynamics). As a result, antibodies with controlled plasma pharmacokinetics can exhibit superior anti-tumor effects, for example, against cancer cells, compared to antibodies with uncontrolled plasma pharmacokinetics.

In the method of the present invention, "maintaining antigen binding activity" means having an activity of at least 80% or more, preferably 85% or more, and particularly preferably 90% or more, compared to the binding activity of the peptide before modification. In addition, when the antibody binds to the antigen, it is sufficient to maintain the activity to the extent that the function possessed by the antibody is maintained. For example, the affinity measured at 37°C under physiological conditions is 100 nM or less, preferably 50 nM or less, more preferably 10 nM or less, and even more preferably 1 nM or less. Whether the polypeptide comprising the antibody variable region with a modified isoelectric point obtained by the method of the present invention maintains antigen binding activity can be determined by known methods, such as BIACORE (molecular interaction analysis), cell proliferation assay, ELISA (enzyme-linked immunosorbent assay), EIA (enzyme immunoassay), RIA (radioimmunoassay), or fluorescent immunoassay.

Examples of the "polypeptide comprising an antibody variable region" of the present invention include antibodies, small molecule antibodies, scaffold proteins, etc., but are not limited to these.

In the present invention, any scaffold protein can be used as long as it is a peptide that can bind to at least one antigen and has a stable three-dimensional structure. Examples of such peptides include antibody variable region fragments, fibronectin, protein A domains, LDL receptor A domains, lipocalins, and the like. In addition, there are molecules described by Nygren et al. (Current Opinion in Structural Biology, (1997) 7:463-469; Journal of Immunol Mothods, 290:3-28 (2004)), Binz et al. (Nature Biotech. 23:1257-1266 (2005)), and Hosse et al. (Protein Science 15:14-27 (2006)).

In the present invention, the term "antibody" is used in its broadest sense, and includes monoclonal antibodies, polyclonal antibodies, antibody mutants (chimeric antibodies, humanized antibodies, small molecule antibodies (including antibody fragments), multispecific antibodies, etc.), as long as they exhibit the desired biological activity. In the present invention, when obtaining (producing) the above-mentioned antibodies, the antibody modification method of the present invention can be preferably used.

The "antibodies" of the present invention include antibodies whose amino acid residue charges have been modified as described above, wherein the amino acid sequence has been further modified by amino acid substitution, deletion, addition, and/or insertion. Antibodies whose amino acid sequence has been modified by amino acid substitution, deletion, addition, and/or insertion, or by chimerization or humanization, wherein the amino acid residue charges have been further modified. In other words, mouse antibodies can be modified simultaneously with humanization, or humanized antibodies can be further modified.

Amino acid sequence modifications such as amino acid substitution, deletion, addition and/or insertion, as well as humanization and chimerization, can be performed according to methods well known to those skilled in the art. Similarly, when the antibodies of the present invention are prepared as recombinant antibodies, the variable and constant regions of the antibodies used can also be modified in their amino acid sequences by amino acid substitution, deletion, addition and/or insertion, or chimerization and humanization.

The antibodies of the present invention may be antibodies derived from any animal, such as mouse antibodies, human antibodies, rat antibodies, rabbit antibodies, goat antibodies, and camel antibodies. Furthermore, they may be modified antibodies with amino acid sequence substitutions, such as chimeric antibodies and humanized antibodies. Furthermore, they may be any antibody, such as modified antibodies bound to various molecules, antibody fragments, and small molecule antibodies.

"Chimeric antibodies" are antibodies produced by combining sequences from different animals. Examples include antibodies composed of the heavy and light chain variable (V) regions of a mouse antibody and the heavy and light chain constant (C) regions of a human antibody. The production of chimeric antibodies is well known. For example, DNA encoding the antibody V region and DNA encoding the human antibody C region are linked, integrated into an expression vector, and then introduced into a host to produce chimeric antibodies.

In addition, the small molecule antibodies in the present invention are not limited to their structure, production method, etc. as long as they have antigen binding ability. Among small molecule antibodies, there are antibodies with higher activity than full-length antibodies (Orita et al., Blood, (2005) 105: 562-566). In this specification, "small molecule antibody" is not particularly limited as long as it is a part of a full-length antibody (such as a full-length IgG, etc.), but preferably contains a heavy chain variable region (VH) or a light chain variable region (VL). Examples of preferred antibody fragments include: Fab, F(ab') ₂ , Fab', Fv, etc. The amino acid sequence of VH or VL in the antibody fragment can be modified by substitution, deletion, addition and/or insertion. Moreover, as long as the antigen binding ability is maintained, part of VH and VL can be deleted. For example, among the above-mentioned antibody fragments, "Fv" is the smallest antibody fragment that contains a complete antigen recognition site and binding site. "Fv" is a dimer (VH-VL dimer) in which one VH and one VL are strongly bound by non-covalent bonds. An antigen-binding site is formed on the surface of the VH-VL dimer by the three complementarity-determining regions (CDRs) of each variable region. The six CDRs confer the binding site between the antigen and the antibody. However, even with a single variable region (or half of an Fv comprising only three antigen-specific CDRs), although its affinity is lower than that of the full binding site, it still has the ability to recognize and bind to the antigen. Therefore, such molecules smaller than Fv are also included in the small molecule antibodies of the present invention. The variable regions of small molecule antibodies can be chimerized or humanized.

Small molecule antibodies preferably include both VH and VL. Examples of small molecule antibodies include antibody fragments such as Fab, Fab', F(ab')2, and Fv, as well as scFv (single-chain Fv) that can be prepared using antibody fragments (Huston et al., Proc. Natl. Acad. Sci. USA (1988) 85: 5879-83; Pluckthun, "The Pharmacology of Monoclonal Antibodies," Vol. 113, Resenburg and Moore (eds.), Springer-Verlag, New York, pp. 269-315, (1994)), diabodies (Holliger et al., Proc. Natl. Acad. Sci. USA (1993) 90: 6444-8; EP 404097; WO 93/11161; Johnson et al., Method in Enzymology (1991) 203: 88-98; Holliger et al., Protein Engineering (1996) 9: 299-305; Perisic et al., Structure (1994) 2: 1217-26; John et al., Protein Engineering (1999) 12 (7): 597-604; Atwell et al., Mol. Immunol. (1996) 33: 1301-12), sc(Fv)2 (Hudson et al., J Immunol. Methods (1999) 231: 177-89; Orita et al., Blood (2005) 105: 562-566), Triabodies (Journal of Immunological Methods (1999) 231: 177-89) and Tandem Diabody (tandem diabody) (Cancer Research (2000) 60: 4336-41), etc.

Antibody fragments can be obtained by treating antibodies with enzymes such as papain, pepsin, and the like (see Morimoto et al., J. Biochem. Biophys. Methods (1992) 24:107-17; Brennan et al., Science (1985) 229:81). Alternatively, they can be produced by genetic recombination based on the amino acid sequence of the antibody fragment.

Small antibodies with modified antibody fragment structures can be constructed using antibody fragments obtained by enzyme treatment or genetic recombination. Alternatively, a gene encoding the entire small antibody can be constructed, introduced into an expression vector, and then expressed in an appropriate host cell (see, for example, Co et al., J. Immunol. (1994) 152:2968-76; Better and Horwitz, Methods Enzymol. (1989) 178:476-96; Pluckthun and Skerra, Methods Enzymol. (1989) 178:497-515; Lamoyi, Methods Enzymol. (1986) 121:652-63; Rousseaux et al., Methods Enzymol. (1986) 121:663-9; Bird and Walker, Trends Biotechnol. (1991) 9:132-7).

The above-mentioned "scFv" is a single-chain polypeptide formed by connecting two variable regions via a linker as needed. The two variable regions contained in scFv are usually one VH and one VL, but can also be two VH or two VL. Generally, the scFv polypeptide contains a linker between the VH and VL domains, through which the paired parts of VH and VL necessary for antigen binding are formed. Generally, in order to form paired parts between VH and VL in the same molecule, the linker connecting VH and VL is usually a peptide linker with a length of more than 10 amino acids. However, the linker of scFv in the present invention is not limited to such a peptide linker as long as it does not hinder the formation of scFv. As a review of scFv, reference can be made to Pluckthun "The Pharmacology of Monoclonal Antibody", Volume 113 (Rosenburg and Moore, Springer Verlag, NY, pp. 269-315 (1994)).

"Diabodies (Db)" refer to bivalent antibody fragments constructed by gene fusion (P. Holliger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993); EP 404,097; WO 93/11161, etc.). Diabodies are dimers composed of two polypeptide chains, each containing a light chain variable region (VL) and a heavy chain variable region (VH) linked by a linker that is so short that they cannot bind to each other, for example, about 5 residues. The VL and VH encoded on the same polypeptide chain cannot form a single-chain V region fragment due to the short linker between them, but instead form a dimer, resulting in a diabody having two antigen-binding sites. In this case, if VL and VH corresponding to two different epitopes (a, b) are simultaneously expressed as a combination of VLa-VHb and VLb-VHa connected by a linker of about 5 residues, they are secreted as bispecific Db.

Because diabodies consist of two scFv molecules, they contain four variable regions and, as a result, have two antigen-binding sites. Unlike scFvs that do not form dimers, when forming diabodies, the linker connecting the VH and VL molecules within each scFv molecule is typically a peptide linker of approximately 5 amino acids. However, the linker used in the scFv forming diabodies is not limited to such peptide linkers, as long as it does not interfere with scFv expression or diabody formation.

Among the multiple isotypes of antibodies, IgG antibodies are not primarily excreted by the kidneys because their molecular weight is large enough. IgG antibodies with an Fc region as part of their molecules are known to be recycled through a salvage pathway of the fetal Fc receptor (FcRn) expressed in endothelial cells of blood vessels, etc., thereby having a longer in vivo half-life. IgG antibodies are believed to be primarily metabolized through metabolic pathways in endothelial cells (He XY, Xu Z, Melrose J, Mullowney A, Vasquez M, Queen C, Vexler V, Klingbeil C, Co MS, Berg EL. Humanization and pharmacokinetics of a monoclonal antibody with specificity for both E- and P-selectin (humanization and pharmacokinetics of E and P-selectin-specific monoclonal antibodies). J Immunol. (1998) 160 (2): 1029-35). That is, it is believed that IgG antibodies that nonspecifically enter endothelial cells are recycled by binding to FcRn, while IgG antibodies that fail to bind are metabolized. IgG antibodies whose Fc regions are modified to reduce their FcRn-binding activity have a shortened plasma half-life. Conversely, by modifying the amino acid residues that constitute the Fc region of an IgG antibody to enhance its FcRn-binding activity, its plasma half-life can be prolonged (He XY, Xu Z, Melrose J, Mullowney A, Vasquez M, Queen C, Vexler V, Klingbeil C, Co MS, Berg EL. Humanization and pharmacokinetics of a monoclonal antibody with specificity for both E- and P-selectin. J Immunol. (1998) 160(2): 1029-35). As described above, conventional methods for controlling the plasma pharmacokinetics of IgG antibodies involve modifying the FcRn-binding activity by modifying the amino acid residues that constitute the Fc region. However, as shown in the Examples below, the present invention has clarified that the plasma half-life of antibodies is highly correlated with the pI. Specifically, the present invention's research has shown that the plasma half-life of antibodies can be controlled without modifying the amino acid sequence constituting the Fc region, and that such modifications have the potential to enhance immunogenicity.

Although not wanting to be bound by a specific theory, the present inventors are thinking as follows at this stage. It is believed that the speed at which IgG antibodies non-specifically enter endothelial cells depends on the physicochemical Coulomb interaction (Coulomb interaction) between the cell surface with a negative charge and the IgG antibody. Therefore, by reducing (increasing) the pI of IgG, the Coulomb interaction is reduced (increased), and the amount of the antibody non-specifically entering the endothelial cells is reduced (increased). As a result, by reducing (increasing) the metabolism of the antibody in the endothelial cells, the plasma pharmacokinetics of the antibody can be controlled. The Coulomb interaction between endothelial cells and the negative charge on the cell surface belongs to a physicochemical interaction, so it is believed that the interaction does not depend on the amino acid sequence itself constituting the antibody. Therefore, the method for controlling the pharmacokinetics in plasma found in the present invention is not only applicable to specific antibodies, but can be widely applied to any polypeptide comprising an antibody variable region. As such a peptide, a peptide with a molecular weight of 50,000 or more is preferred, a peptide with a molecular weight of 100,000 or more is more preferred, and a peptide with a molecular weight of 140,000 or more is further preferred. If such a peptide, its main metabolic pathway is not renal excretion, and the plasma pharmacokinetic control effect of the present invention can be fully obtained. It should be noted that the reduction (increase) of the Coulomb interaction in the present invention refers to the increase (decrease) of the Coulomb force expressed as a repulsive force.

The polypeptide comprising an FcRn-binding region of the present invention is not limited to an IgG antibody; any protein capable of binding to (having binding activity or affinity for) an Fc receptor (FcRn) may be used. The polypeptide comprising an FcRn-binding region of the present invention is not particularly limited, but is preferably a protein comprising an antibody Fc region or Fc-like region. A modified Fc region may also be used, for example, the modified Fc region described in J Immunol. (1998) 160(2):1029-35. Examples of polypeptides comprising an FcRn-binding region of the present invention include IgG antibodies. Furthermore, even modified forms of these antibodies (proteins) are encompassed by the polypeptide comprising an FcRn-binding region of the present invention as long as they are proteins capable of binding to FcRn. The most preferred example of a polypeptide comprising an FcRn-binding region of the present invention is an IgG antibody.

When using IgG antibody as antibody of the present invention, as long as it is IgG type antibody molecule, it can be any subtype, or it can be a multi-specific (such as bispecific) IgG antibody. This bispecific antibody is an antibody with specificity to two different epitopes, and except comprising the antibody that identifies different antigens, it also comprises the antibody that identifies the different epitopes on the same antigen. In addition, even if it is an antibody molecule, when it is a small molecule antibody such as scFv or Fab with renal excretion as the main metabolic pathway, as mentioned above, by controlling pI, it is also not possible to control pharmacokinetics in plasma, but as long as it is not with renal excretion as the main metabolic pathway, comprising the polypeptide of antibody variable region, then no matter any form of antibody molecule the present invention all can be applicable, such polypeptide for example has: scFv-Fc, dAb-Fc, Fc fusion protein etc. Because these molecules are not with renal excretion as the main metabolic pathway, so by utilizing the method found in the present invention to change pI, it is possible to control pharmacokinetics in plasma. The antibody molecule that the present invention can be applicable to can be an antibody-like molecule. "Antibody-like molecules" refer to molecules that function by binding to target molecules (Binz HK, Amstutz P, Pluckthun A. Engineering novel binding proteins from nonimmunoglobulin domains. Nat Biotechnol. 2005 Oct; 23(10): 1257-68), including, for example, DARPins, Affibodies, Avimers, etc.

It should be noted that when the antibody of the present invention is, for example, a bispecific anti-glypican 3 antibody, the antibody can also specifically bind to glypican 3 and antigenic epitopes other than glypican 3. As antigens other than glypican 3, for example, in order to recruit NK cells, cytotoxic T cells, LAK cells, etc., surface antigens that specifically bind to these cells can be preferably used. Studies have shown that a bispecific antibody prepared from the antibody MUSE11, which recognizes the adenocarcinoma-associated antigen MUC1, and the antibody OKT3, which recognizes the LAK cell surface antigen, can exert cytotoxicity against cholangiocarcinoma by LAK cells (Katayose Y, Kudo T, Suzuki M, Shinoda M, Saijyo S, Sakurai N, Saeki H, Fukuhara K, Imai K, Matsuno S. MUC1-specific targeting immunotherapy with bispecific antibodies: inhibition of xenografted human bile duct carcinoma growth. Cancer Res. (1996) 56(18): 4205-12). The glypican 3 antibody provided by the present invention, which has improved plasma pharmacokinetics, can preferably be used instead of the MUC1-recognizing antibody MUSE11. As the bispecific glypican 3 antibody provided by the present invention, antibodies that recognize different epitopes of the glypican 3 molecule can also be preferably used.

It should be noted that the above-mentioned "bispecific antibody" can be, for example, an antibody having a structure in which the heavy chain variable region and the light chain variable region are connected in a single chain form (e.g., sc(Fv)2). It can also be an antibody-like molecule (e.g., scFv-Fc) obtained by connecting the heavy chain variable region (VH) and the light chain variable region (VL) to an Fc region (a constant region lacking the CH1 domain). The multispecific antibody formed by scFv-Fc has a (scFv)2-Fc structure, wherein the first polypeptide is VH1-linker-VL1-Fc and the second polypeptide is VH2-linker-VL2-Fc. Alternatively, it can be an antibody-like molecule obtained by combining a single domain antibody with an Fc region (Curr. Opin. Drug Discov. Devel. (2006) 9(2):184-93).

In the present invention, the charge of an amino acid residue can be altered by amino acid substitution. Amino acid substitution can be performed according to the method described below.

It should be noted that, in the present invention, the amino acid residues that can be exposed on the surface of the CDR region as the subject of substitution are preferably selected from the group consisting of amino acid residues at positions 31, 61, 62, 64, and 65 in the heavy chain variable region according to Kabat numbering, or amino acid residues at positions 24, 27, 53, 54, and 55 in the light chain variable region according to Kabat numbering, from the perspective of maintaining antigen-binding activity. Substitution of these amino acid residues is useful in maintaining the function (antigen-binding activity, etc.) possessed by the polypeptide comprising the antibody variable region before the amino acid residue substitution, and in that the substitution is independent of the antibody species.

The present invention also provides a method for controlling the pharmacokinetics of a polypeptide comprising an antibody variable region by altering the isoelectric point of the polypeptide. Furthermore, the polypeptide comprising an antibody variable region whose pharmacokinetics are controlled and obtained by this method is also encompassed by the present invention.

In the present invention, "controlling plasma pharmacokinetics" means that the plasma pharmacokinetics of the antibody before and after modification of the amino acids constituting the antibody are compared and that the plasma pharmacokinetics are altered in the desired direction. Specifically, when it is desired to extend the antibody's drug (plasma) half-life, "controlling plasma pharmacokinetics" means extending the antibody's plasma half-life. When it is desired to shorten the antibody's plasma half-life, "controlling plasma pharmacokinetics" means shortening the antibody's plasma half-life.

In the present invention, whether the pharmacokinetics of the antibody in plasma changes in the desired direction, that is, whether the pharmacokinetics in plasma is controlled as desired, can be appropriately evaluated by performing kinetic tests using, for example, mice, rats, rabbits, dogs, monkeys, etc. More specifically, in addition to using the parameter half-life (t1/2) in plasma for evaluation, the "prolongation of half-life in plasma" or "shortening of half-life in plasma" in the present invention can also be grasped by any one of the parameters such as mean plasma residence time, plasma clearance (CL), and area under the concentration curve (AUC) ("Pharmacokinetics practice による understanding" (Nanshan Hall)). For example, non-compartmental analysis is performed according to the operating procedures included in the in vivo kinetic analysis software WinNonlin (Pharsight), so that these parameters can be used to appropriately evaluate the "control of kinetics in plasma" provided by the present invention.

By controlling the pharmacokinetics in plasma, the antibody's function can be sustained. For example, when the methods of the present invention are applied to cytotoxic antibodies, their function can be sustained, and the duration of the pre-modified polypeptide's function, such as cytotoxicity, antagonism, or agonism, can be modulated.

In the present invention, "amino acid residues capable of being exposed to the surface" generally refers to amino acid residues located on the surface of the polypeptide constituting the antibody. "Amino acid residues located on the surface of the polypeptide" refers to amino acid residues whose side chains can be in contact with solvent molecules (usually mostly water molecules). It is not necessarily required that all of its side chains are in contact with solvent molecules. Even in the case where a part of its side chains are in contact with solvent molecules, its amino acid residue is defined as "amino acid located on the surface". By using homology modeling methods (homology modeling) using commercially available software, those skilled in the art can prepare homology modeling of polypeptides or antibodies. According to this homology modeling, amino acid residues located on the surface of the polypeptide constituting an appropriate antibody can be appropriately selected as "amino acid residues located on the surface of the polypeptide".

In the present invention, "surface-exposed amino acid residues" are not particularly limited, but are preferably amino acid residues located outside the FcRn-binding region of an antibody. Preferred examples of such FcRn-binding regions include the Fc region.

In the antibodies of the present invention, the amino acid residues whose charge is to be altered are preferably amino acid residues constituting the heavy chain variable region or light chain variable region of the antibody. Specific preferred examples of such variable regions include complementarity determining regions (CDRs) and framework regions (FRs).

As for the surface amino acid residues in the antibody variable region, those skilled in the art can appropriately select them using homology modeling prepared by homology modeling, etc. That is, the surface amino acid residues in the antibody variable region can be appropriately selected from the amino acid residues H1, H3, H5, H8, H10, H12, H13, H15, H16, H19, H23, H25, H26, H31, H39, H42, H43, H44, H46, H61, H62, H64, H65, H68, H71, H72, H73, H75, H76, H81, H82b, H83, H85, H86, H105, H108, H110, and H112 according to the Kabat numbering of the H chain variable region. For example, in the H chain FR region of the humanized Glypican 3 antibody shown in SEQ ID NO: 195, surface amino acids can be exemplified by amino acid residues at positions 1, 3, 5, 8, 10, 12, 13, 15, 16, 19, 23, 25, 26, 39, 42, 43, 44, 46, 69, 72, 73, 74, 76, 77, 82, 85, 87, 89, 90, 107, 110, 112, and 114, but the present invention is not limited thereto. In addition, surface amino acid residues in the H chain CDR can be selected based on the same homology modeling. That is, amino acid residue H97 according to Kabat numbering is exposed on the surface of almost all antibodies. For example, the serine at position 101 in the H chain CDR of the humanized Glypican 3 antibody shown in SEQ ID NO: 195 corresponds to this amino acid residue. Preferred examples of other amino acid residues in the H-chain CDR of the humanized Glypican 3 antibody represented by SEQ ID NO: 195 include amino acid residues at positions 52, 54, 62, 63, 65, and 66.

In the L chain variable region, the surface amino acid residues in the antibody variable region can be appropriately selected from amino acid residues L1, L3, L7, L8, L9, L11, L12, L16, L17, L18, L20, L22, L24, L27, L38, L39, L41, L42, L43, L45, L46, L49, L53, L54, L55, L57, L60, L63, L65, L66, L68, L69, L70, L74, L76, L77, L79, L80, L81, L85, L100, L103, L105, L106, and L107 according to Kabat numbering. For example, 1, 3, 7, 8, 9, 11, 12, 16, 17, 18, 20, 22, 43, 44, 45, 46, 48, 49, 50, 54, 62, 65, 68, 70, 71, 73, 74, 75, 79, 81, 82, 84, 85, 86, 90, 105, 108, 110, 111, and 112 of the humanized Glypican 3 antibody shown in SEQ ID NO: 195 can be exemplified as surface amino acids, but the present invention is not limited to these. Thus, as for the surface amino acid residues in the L chain CDR, they can be selected based on the same homology modeling as that used to determine the surface amino acid residues in the H chain CDR. As amino acid residues in the L chain CDR of the humanized Glypican 3 antibody shown in SEQ ID NO: 201, preferably, amino acid residues at positions 24, 27, 33, 55, and 59 can be mentioned.

In the methods provided herein, "modification" of an amino acid residue specifically refers to substitution of an original amino acid residue with another amino acid residue, deletion of an original amino acid residue, addition of a new amino acid residue, etc., but preferably refers to substitution of an original amino acid residue with another amino acid residue. That is, in the present invention, "alteration of the charge of an amino acid residue" preferably refers to amino acid substitution.

In the glypican 3 antibodies provided by the present invention, in order to perform the above-mentioned "alteration of the charge of amino acid residues," it is preferred that the charge of at least one amino acid residue selected from amino acid residues 19, 43, 52, 54, 62, 63, 65, 66, and 107 in the H chain variable region of the humanized glypican 3 antibody as shown in SEQ ID NO: 195 be altered. For example, it is preferred that the charge of at least one amino acid residue selected from amino acid residues 17, 24, 27, 33, 55, 59, 79, 82, and 105 in the L chain variable region of the humanized glypican 3 antibody as shown in SEQ ID NO: 201 be altered. Among the above-mentioned amino acid residues, amino acid residues other than the amino acid residues whose charge has been altered do not need to be modified as long as the desired effect of controlling plasma pharmacokinetics is achieved. However, they may be appropriately modified so that they have the same charge as the appropriately modified amino acid residue or have no charge.

In the CDRs of the anti-human IL-6 receptor antibody (6R_a_H1L1) provided herein, in order to modify the "charge of the amino acid residue" while maintaining antigen-binding activity, it is preferred to modify the charge of at least one amino acid residue selected from amino acid residues 31, 64, and 65 by Kabat numbering in the H chain variable region of the anti-human IL-6 receptor antibody as set forth in SEQ ID NO: 221. Furthermore, it is preferred to modify the charge of at least one amino acid residue selected from amino acid residues 24, 27, 53, and 55 by Kabat numbering in the L chain variable region of the anti-human IL-6 receptor antibody as set forth in SEQ ID NO: 224. Among the above amino acid residues, amino acid residues other than the amino acid residue whose charge has been modified do not need to be modified as long as the desired effect of controlling plasma pharmacokinetics is achieved. However, these residues may be appropriately modified to have the same charge as the appropriately modified amino acid residue or to have no charge.

In the CDRs of the anti-human IL-6 receptor antibody (6R_b_H1L1) provided herein, in order to modify the "charge of the amino acid residue" while maintaining antigen-binding activity, it is preferred to modify the charge of at least one amino acid residue selected from the group consisting of amino acid residue 31 by Kabat numbering in the H chain variable region of the anti-human IL-6 receptor antibody as set forth in SEQ ID NO: 227. Furthermore, it is preferred to modify the charge of at least one amino acid residue selected from the group consisting of amino acid residues 24, 53, 54, and 55 by Kabat numbering in the L chain variable region of the anti-human IL-6 receptor antibody as set forth in SEQ ID NO: 229. Among the above amino acid residues, amino acid residues other than the amino acid residue whose charge has been modified do not need to be modified as long as the desired effect of controlling plasma pharmacokinetics is achieved. However, they may be appropriately modified to have the same charge as the appropriately modified amino acid residue or to have no charge.

In the CDRs of the anti-human GPC3 antibodies provided herein, in order to modify the charge of the amino acid residues described above while maintaining antigen-binding activity, it is preferred to modify the charge of at least one amino acid residue selected from amino acid residues 61, 62, 64, and 65 in the H chain variable region of the anti-human GPC3 antibody as shown in SEQ ID NO: 233, as defined by Kabat numbering. Furthermore, it is preferred to modify the charge of at least one amino acid residue selected from amino acid residues 24 and 27 in the L chain variable region of the anti-human GPC3 antibody as defined in SEQ ID NO: 236, as defined by Kabat numbering. Among the above amino acid residues, amino acid residues other than the amino acid residues whose charge has been modified do not need to be modified as long as the desired effect of controlling plasma pharmacokinetics is achieved. However, these residues may be appropriately modified to have the same charge as the appropriately modified amino acid residue or to have no charge.

In the CDRs of the anti-human IL-31 receptor antibodies provided herein, in order to modify the "charge of the amino acid residue" while maintaining antigen-binding activity, it is preferred to modify the charge of at least one amino acid residue selected from amino acid residues 61, 62, 64, and 65 (Kabat numbering) in the H chain variable region of the anti-human IL-31 receptor antibody as set forth in SEQ ID NO: 239. Furthermore, it is preferred to modify the charge of at least one amino acid residue selected from amino acid residues 24 and 54 (Kabat numbering) in the L chain variable region of the anti-human IL-31 receptor antibody as set forth in SEQ ID NO: 242. Among the above amino acid residues, amino acid residues other than the amino acid residues whose charge has been modified do not need to be modified as long as the desired effect of controlling plasma pharmacokinetics is achieved. However, these residues may be appropriately modified to have the same charge as the appropriately modified amino acid residue or to have no charge.

It is known that there are charged amino acids among amino acids. Generally, lysine (K), arginine (R), and histidine (H) are known as positively charged amino acids (positively charged amino acids). Aspartic acid (D), glutamic acid (E) and the like are known as negatively charged amino acids (negatively charged amino acids). Other amino acids are known as uncharged amino acids.

The "modified amino acid residue" is preferably selected from the amino acid residues included in either group (a) or (b) below, but is not particularly limited to these amino acids.

(a) Glutamic acid (E), aspartic acid (D);

(b) Lysine (K), Arginine (R), Histidine (H)

It should be noted that when the original (pre-modification) amino acid residue already carries a charge, it can be modified to become an uncharged amino acid residue, which is also one of the preferred embodiments of the present invention. Specifically, the modifications in the present invention include: (1) substituting a charged amino acid for an uncharged amino acid; (2) substituting a charged amino acid for an amino acid with an opposite charge; and (3) substituting an uncharged amino acid for a charged amino acid.

In the present invention, it is preferred to modify the amino acid residues constituting the antibody to change the isoelectric point (pI) of the antibody. When there are multiple amino acid residues to be modified, a small number of uncharged amino acid residues may be included in the amino acid residues for modification.

In the Glypican 3 antibody provided by the present invention, preferred examples of "alterations of the charge of amino acid residues" are listed below. As modifications to increase the pI value, for example, at least one of Q43K, D52N, and Q107R in the H chain variable region of the humanized Glypican 3 antibody shown in SEQ ID NO: 195 can be substituted, and modification to the amino acid sequence shown in SEQ ID NO: 198 is particularly preferred. Furthermore, for example, at least one of E17Q, Q27R, and Q105R in the L chain variable region of the humanized Glypican 3 antibody shown in SEQ ID NO: 201 can be substituted, and modification to the amino acid sequence shown in SEQ ID NO: 204 is particularly preferred. On the other hand, as a modification to reduce the pI value, at least one of K19T, Q43E, K63S, K65Q, and G66D in the H chain variable region constituting the humanized Glypican 3 antibody shown in SEQ ID NO: 195 can be substituted, and particularly preferably modified to the amino acid sequence shown in SEQ ID NO: 197. Furthermore, for example, at least one of Q27E, K79T, and R82S in the L chain variable region constituting the humanized Glypican 3 antibody shown in SEQ ID NO: 201 can be substituted, and particularly preferably modified to the amino acid sequence shown in SEQ ID NO: 203.

In the anti-human IL-6 receptor antibody (6R_a_H1L1) provided by the present invention, preferred examples of "alteration of the charge of an amino acid residue" include at least one amino acid substitution listed in Table 20.

In the anti-human IL-6 receptor antibody (6R_b_H1L1) provided by the present invention, preferred examples of "alteration of the charge of an amino acid residue" include at least one amino acid substitution listed in Table 22.

In the anti-human GPC3 antibody provided by the present invention, preferred examples of "alteration of the charge of an amino acid residue" include at least one amino acid substitution among the amino acid substitutions listed in Table 24.

In the anti-human IL-31 receptor antibody provided by the present invention, preferred examples of "alteration of the charge of an amino acid residue" include at least one amino acid substitution listed in Table 27.

In the present invention, the number of amino acid residues to be modified is not particularly limited. However, when modifying an antibody variable region, for example, in order to avoid reducing antigen-binding activity and increasing immunogenicity, it is preferred to modify the minimum amino acid residues necessary to achieve the desired controlled plasma pharmacokinetics. It is also preferred to appropriately combine modifications of amino acid residues that enhance antigen-binding activity with modifications of amino acid residues that reduce immunogenicity.

The binding activity between an antibody and an antigen can be measured using known methods. For example, ELISA (enzyme-linked immunosorbent assay), EIA (enzyme immunoassay), RIA (radioimmunoassay), or fluorescent immunoassay can be used. These methods are described in the general textbook "Antibodies: A Laboratory Manual. Ed Harlow, David Lane, Cold Spring Harbor Laboratory, 1988."

As a method for measuring the binding activity of antibodies to cells, there is, for example, the method described on pages 359 to 420 of Antibodies A Laboratory Manual. (Ed Harlow, David Lane, Cold Spring Harbor Laboratory, 1988). That is, the activity can be evaluated according to the principle of BIACORE, cell proliferation analysis, ELISA or FACS (fluorescence activated cell sorting) using cells as antigens. In the ELISA method, the binding activity of antibodies to cells is quantitatively evaluated by comparing the signal levels generated by the enzyme reaction. That is, the antibody to be tested is added to the ELISA plate fixed with each forced expression cell, and the antibody bound to the cell is detected using an enzyme-labeled antibody that recognizes the antibody to be tested. Alternatively, in FACS, a dilution series of the antibody to be tested is made, and the binding titer of the antibody to each forced expression cell is determined, and the binding activity of the antibody to the cell can be compared.

The binding of an antigen expressed on the surface of cells suspended in a buffer solution, not bound to a support such as an ELISA plate, and an anti-antigen antibody can be measured using FACS. Examples of flow cytometers used in such measurements include the FACSCanto ^™ II, FACSAria ^™ , FACSArray ^™ , FACSVantage ^™ SE, and FACSCalibur ^™ (all from BD Biosciences), as well as the EPICS ALTRAHyPerSort, Cytomics FC500, EPICS XL-MCL ADC, EPICS XL ADC, and Cell Lab Quanta/Cell Lab Quanta SC (all from Beckman Coulter).

As an example of a preferred assay method for the binding activity of an antibody to an antigen, the following method can be cited: cells expressing an antigen are reacted with a test antibody, the cells are stained with a secondary antibody that recognizes the test antibody and is FITC-labeled, and then FACSCalibur (BD) is used to measure the fluorescence intensity, and CELL QUEST software (BD) is used to analyze the method. According to this method, cells are stained with a secondary antibody that specifically recognizes the test antibody and is bound to an antigen on the surface of cells expressing an antigen and is FITC-labeled, and then FACSCalibur is used to measure the fluorescence intensity, and the geometric mean (tested Geo-Mean value) obtained by the method is compared with the control Geo-Mean value obtained using a control antibody, so as to be able to judge. The formula for calculating the Geo-Mean value (geometric mean) is described in the CELL QUEST software user guide (BD Biosciences).

In order not to increase the in vivo immunogenicity of the human to whom the antibody is to be administered, the modified amino acid sequence is preferably a human sequence (a sequence found in natural human antibodies), but the present invention is not limited thereto. Furthermore, in order to convert multiple FRs (FR1, FR2, FR3, FR4) into human sequences after modification, it is preferable to introduce mutations into sites other than the sites into which the modification has been introduced in order to change the isoelectric point. The method of replacing each FR with a human sequence in this manner is reported in the non-patent literature (Ono K, Ohtomo T, Yoshida K, Yoshimura Y, Kawai S, Koishihara Y, Ozaki S, Kosaka M, Tsuchiya M. The humanized anti-HM1.24 antibody effectively kills multiple myeloma cells by human effector cell-mediated cytotoxicity. Mol. Immunol. (1999) 36(6): 387-395). In addition, to change the isoelectric point of the antibody, each FR can be modified with another human FR to change its charge (for example, FR3 can be exchanged with another human FR to lower the isoelectric point of the antibody). Such humanization methods are reported in non-patent literature (Dall'Acqua WF, Damschroder MM, Zhang J, Woods RM, Widjaja L, Yu J, Wu H. Antibody humanization by framework shuffling. Methods. (2005) 36 (1): 43-60).

In addition, when the target controlled plasma pharmacokinetics cannot be achieved by only a slight change in surface charge, the desired antibody with controlled target plasma pharmacokinetics can be preferably obtained by repeatedly performing changes in surface charge and evaluating plasma pharmacokinetics.

In a non-patent document (He XY, Xu Z, Melrose J, Mullowney A, Vasquez M, Queen C, Vexler V, Klingbeil C, Co MS, Berg EL. Humanization and pharmacokinetics of a monoclonal antibody with specificity for both E- and P-selectin. J Immunol. (1998) 160(2): 1029-35), the pharmacokinetics of an anti-E, P-selectin chimeric antibody (IgG4), i.e., chimeric EP5C7.g4, and a humanized antibody (IgG4), i.e., HuEP5C7.g4, in plasma in macaques were compared. The results showed that the pharmacokinetics of the two antibodies in plasma were equivalent. In the non-patent literature (Gobburu JV, Tenhoor C, Rogge MC, Frazier DE Jr, Thomas D, Benjamin C, Hess DM, Jusko WJ. Pharmacokinetics/dynamics of 5c8, a monoclonal antibody to CD154 (CD40 ligand) suppression of an immune response in monkeys. J Pharmacol Exp Ther. (1998) 286(2): 925-30), the pharmacokinetics of the anti-CD154 chimeric antibody ch5d8 and the humanized antibody Hu5c8 in plasma in cynomolgus monkeys were compared, and the results showed that the pharmacokinetics of the two antibodies in plasma were equivalent. A non-patent document (Kashmiri SV, Shu L, Padlan EA, Milenic DE, Schlom J, Hand PH., Generation, characterization, and in vivo studies of humanized anticarcinoma antibody CC49. Hybridoma. (1995) 14(5): 461-73) shows that the chimeric antibody cCC49 and the humanized antibody HuCC49 have equivalent pharmacokinetics in plasma in mice. Non-patent literature (Graves SS, Goshorn SC, Stone DM, Axworthy DB, Reno JM, Bottino B, Searle S, Henry A, Pedersen J, Rees AR, Libby RT. Molecular modeling and preclinical evaluation of the humanized NR-LU-13 antibody. Clin Cancer Res. (1999) 5(4): 899-908) and non-patent literature (Couto JR, Blank EW, Peterson JA, Ceriani RL. Anti-BA46 monoclonal antibody Mc3: humanization using a novel positional consensus and in vivo and in vitro characterization. Cancer Res. (1995) 55(8): 1717-22) show that in an evaluation in mice, the pharmacokinetics and distribution in plasma of mouse antibodies were equivalent to those of humanized antibodies. It is believed that since both mouse Fc and human Fc cross with mouse FcRn, the plasma pharmacokinetics and distribution of the same chimeric antibody and the same humanized antibody are equivalent. As shown in these examples, the plasma pharmacokinetics of chimeric antibodies and humanized antibodies with the same CDRs are equivalent. That is, when humanized according to the known methods shown in non-patent literature (Ghetie V, Popov S, Borvak J, Radu C, Matesoi D, Medesan C, Ober RJ, Ward ES. Increasing the serum persistence of an IgG fragment by random mutagenesis (using random mutagenesis to improve the serum retention of IgG fragments). Nat Biotechnol. (1997) 15 (7): 637-40), the plasma pharmacokinetics are equivalent to those of chimeric antibodies, and it is not possible to produce humanized antibodies with controlled plasma pharmacokinetics according to the known methods.

In contrast, when chimeric antibodies are humanized using the methods discovered in the present invention, surface-exposed amino acid residues of the chimeric antibody are modified to alter the antibody's pI, thereby producing humanized antibodies with controlled plasma pharmacokinetics (i.e., an increased or decreased plasma half-life) compared to the chimeric antibody. Modification of surface-exposed amino acid residues of the humanized antibody to control plasma pharmacokinetics can be performed simultaneously with antibody humanization, or the pI of the humanized antibody can be further altered by using the humanized antibody as a starting material and modifying surface-exposed amino acid residues.

The value of the isoelectric point in the present invention can be determined by isoelectric point electrophoresis known to those skilled in the art. The value of the theoretical isoelectric point can be calculated using gene and amino acid sequence analysis software (Genetyx, etc.). In the present invention, for example, when the isoelectric point must be significantly changed in order to fully control the pharmacokinetics in plasma, the above-mentioned determination is useful. It is particularly preferred to calculate the value of the theoretical isoelectric point so that the value of the isoelectric point must be changed by more than 1.0, and more preferably, the value of the isoelectric point must be changed by more than 3.0.

A non-patent document (Adams CW, Allison DE, Flagella K, Presta L, Clarke J, Dybdal N, McKeever K, Sliwkowski MX. Humanization of a recombinant monoclonal antibody to produce a therapeutic HER dimerization inhibitor, pertuzumab. Cancer Immunol Immunother. (2006) 55(6):717-27) states that the plasma pharmacokinetics of three humanized antibodies, trastuzumab, bevacizumab, and pertuzumab, humanized using the same human antibody FR sequence are almost identical. In other words, when humanized using the same FR sequence, the plasma pharmacokinetics of the antibodies are almost identical. According to the method discovered in the present invention, in addition to the above-mentioned humanization step, amino acid residues that can be exposed on the antibody surface are modified to change the antibody's pI, thereby controlling the drug (in plasma) concentration.

The methods of the present invention can also be applied to human antibodies. By altering amino acid residues that can be exposed on the surface of human antibodies produced from human antibody libraries or human antibody-producing mice, the pI of the human antibody is altered, and human antibodies can be produced with controlled plasma pharmacokinetics (i.e., prolonged or shortened plasma half-life) compared to the plasma pharmacokinetics of the originally produced human antibody.

By reducing the pI value of an antibody, the half-life of the antibody in plasma is prolonged. Conversely, by increasing the pI value of an antibody, the half-life of the antibody in plasma is shortened and the tissue mobility of the antibody is improved (Vaisitti T, Deaglio S, Malavasi F. Cationization of monoclonal antibodies: another step towards the "magic bullet"?, J Biol Regul Homeost Agents. (2005) 19(3-4): 105-12; Pardridge WM, Buciak J, Yang J, Wu D. Enhanced endocytosis in cultured human breast carcinoma cells and in vivo biodistribution in rats of a humanized monoclonal antibody after cationization of the protein. (1998) 286(1): 548-54). However, due to the increased immunogenicity and intracellular internalization activity of these antibodies, further improvements are needed to adapt them to antibodies that exert cancer therapeutic effects through mechanisms such as cytotoxicity, where internalization activity, such as ADCC and CDC activity, is a key factor in suppressing their activity. Specifically, it is unclear whether an increase or decrease in the pI value enhances tumor suppression in antibodies that exert cancer therapeutic effects through mechanisms such as cytotoxicity, where internalization activity, such as ADCC and CDC activity, is a key factor in suppressing their activity. In the present invention, modified humanized antibodies with reduced and increased pI values were prepared, and their anti-tumor effects were compared to verify whether either modification resulted in a stronger tumor suppression effect. Surprisingly, the humanized antibody with a reduced pI value exhibited a superior effect against liver cancer.

The "antibodies" of the present invention include antibodies whose amino acid residue charges have been modified as described above as a starting material, and whose amino acid sequences have been further modified by substitution, deletion, addition, and/or insertion of amino acid residues constituting the antibody. The "antibodies" of the present invention also include antibodies whose amino acid sequences have been modified by substitution, deletion, addition, and/or insertion of amino acid residues, or by chimerization or humanization as a starting material, and whose amino acid residue charges have been further modified.

As an example of modifications for the purpose of improving the characteristics of the antibodies provided by the present invention, it is preferred to cite: modifications for the purpose of improving antibody stability (hereinafter referred to as "modifications of stability"). Antibodies in aqueous solutions reach equilibrium between the native state and the inert denatured state. As shown in the second law of thermodynamics (ΔG=ΔH-TΔS), the stability of the native state depends on the balance between the change ΔG of the Gibbs free energy of the system and the change ΔH of enthalpy (caused by changes in hydrophobic interactions and hydrogen bonds in the polypeptide chain, etc.) and the change ΔS of entropy (caused by changes in the degree of freedom of the solvent and the three-dimensional structure). A positive ΔG indicates that the native state of the protein is more stable than the denatured state of the protein. The greater the positive value of ΔG, the higher the stability of the native state of the protein. In order for a protein to denature, it is necessary to destroy the force that contributes to its stabilization. For example, by exposing a protein solution to high temperature, the degree of freedom of its three-dimensional structure increases, and the factors that contribute to protein stability are weakened, thereby causing thermal denaturation of the protein, but at this time the -TΔS term determines denaturation. As for the stretched ΔH caused by the thermal denaturation of the protein, it can be directly measured using differential scanning calorimetry (DSC), as specifically described in the examples described in this specification. The DSC curve during the thermal denaturation of the protein forms an endothermic peak at a temperature inherent to the protein under test, called the denaturation midpoint (Tm). This peak is integrated to obtain the change in denaturation enthalpy. The Tm value is usually an indicator of thermal stability. DSC can also be used to measure the change in heat capacity (ΔCp) when the protein undergoes thermal denaturation. The change in heat capacity caused by thermal denaturation is mainly due to the fact that amino acid residues that are not exposed to the molecular surface when the protein exists in its native state are exposed to solvent molecules as the protein denatures, resulting in hydration.

As described above, in the method provided by the present invention, "modification" of an amino acid residue specifically refers to replacing the original amino acid residue with another amino acid residue, deleting the original amino acid residue, adding a new amino acid residue, etc., but preferably replacing the original amino acid residue with another amino acid residue. That is, for the modification of the stability of the antibody in the present invention, it is preferably modified by amino acid substitution. The amino acid residues constituting the antibody are modified for stability, and as a result, the above-mentioned Tm value of the antibody increases. That is, as an indicator of modified antibody stability, it is preferably used the above-mentioned Tm value.

In order to modify the stability of the Glypican 3 antibody provided by the present invention, for example, it is preferred to modify at least one amino acid residue selected from amino acid residues 37, 40, 48, and 51 in the H chain variable region of the humanized Glypican 3 antibody as shown in SEQ ID NO: 195. Furthermore, for example, it is preferred to modify at least one amino acid residue selected from amino acid residues 2, 25, 42, 48, 50, 83, and 84 in the L chain variable region of the humanized Glypican 3 antibody as shown in SEQ ID NO: 201. Among the above amino acid residues, amino acid residues other than those subjected to the stability modification do not need to be modified as long as the desired Tm value is obtained. However, they may be appropriately modified to have a Tm value comparable to or greater than that of the humanized Glypican 3 antibody to be modified.

Modification of stability can be performed by randomly modifying the amino acid residues that constitute the humanized antibody to be modified. Furthermore, modification of stability can also be performed by replacing a portion of the amino acid sequence that constitutes the humanized antibody to be modified with an amino acid sequence that constitutes an existing antibody with a high Tm value and that corresponds to the amino acid sequence of the humanized antibody to be modified from the perspective of the antibody's three-dimensional structure. The position of the amino acid residue to be substituted is not particularly limited, but amino acid residues in the FR region can be preferably modified. Furthermore, even amino acid residues in the CDR region can be appropriately modified as long as they do not result in a decrease in antigen-binding activity. There is no particular limitation on the number of amino acid residues to be modified, and modification can also be performed by replacing a specific segment in the FR region with a desired segment. All segments of FR1, FR2, FR3, and FR4 in the FR region can be modified, and a combination of modifications of one or more segments can also be performed.

When modifying a segment of the FR region, preferred examples include the FR2 region of the H or L chain. Preferred specific examples include, for example, modifying the H chain FR2 of the humanized Glypican 3 antibody with the VH1b subclass, as shown in SEQ ID NO:195, to amino acid residues of the VH4 subclass, i.e., replacing valine at position 37 with isoleucine, V37I; and modifications such as A40P, M48I, and L51I. Preferred specific examples include modifying the L chain FR2 region of the humanized Glypican 3 antibody with the VK2 subclass, as shown in SEQ ID NO:201, to VK3 subclass, i.e., modifying L42Q, S48A, and Q50R; and modification V2I, which corresponds to modification to the germline sequence of FR1.

The substitution, deletion, addition and/or insertion of the amino acid residues constituting the antibody, as well as the modification of the amino acid sequence such as humanization and chimerization, can preferably be performed using methods well known to those skilled in the art. Similarly, when preparing the antibodies provided by the present invention as recombinant antibodies, the substitution, deletion, addition and/or insertion of the amino acid residues constituting the variable and constant regions of the antibody are also preferably performed.

Antibodies used in the present invention are preferably those derived from any animal, such as mouse antibodies, human antibodies, rat antibodies, rabbit antibodies, goat antibodies, and camel antibodies. Furthermore, chimeric antibodies, particularly humanized antibodies, and other modified antibodies in which the amino acid sequence is substituted can also be preferably used. Modified antibodies bound to various molecules can also be preferably used.

"Chimeric antibodies" are antibodies produced by combining sequences from different animals. For example, a preferred example is an antibody composed of the variable (V) regions of the H and L chains of a mouse antibody and the constant (C) regions of the H and L chains of a human antibody. Methods for producing chimeric antibodies are well known. For example, DNA encoding the antibody V region and DNA encoding the human antibody C region are fused in frame, and the resulting recombinant DNA is integrated into a commonly used expression vector. By culturing host cells into which the vector has been introduced, chimeric antibodies can be appropriately obtained or isolated from their culture medium.

"Humanized antibodies", also known as reconstructed human antibodies, are antibodies formed by connecting the complementarity determining regions (CDRs) of antibodies isolated from mammals other than humans, such as mice, to the framework regions (FRs) of human antibodies. The DNA sequence encoding the humanized antibody can be synthesized by overlapping PCR reactions using multiple oligonucleotides as templates. The raw materials for overlapping PCR reactions and their implementation methods are described in WO98/13388, etc. The DNA encoding the variable regions of the humanized antibodies of the present invention is obtained by overlapping PCR reactions with multiple oligonucleotides made into overlapping nucleotide sequences, which are then connected to the DNA encoding the constant regions of the human antibodies in a form that conforms to the reading frame to form a codon sequence. The DNA connected in the above manner is then inserted into an expression vector in a form that enables the DNA to be expressed, and then introduced into the host.

Methods for identifying CDRs are well known (Kabat et al., Sequence of Proteins of Immunological Interest (1987), National Institute of Health, Bethesda, Md.; Chothia et al.; Nature (1989) 342: 877). General gene recombination methods are also well known (see European Patent Application Publication No. EP 125023; WO96/02576). By using the above-mentioned well-known methods, the CDRs of antibodies obtained from non-human animals such as mouse antibodies are determined, and then DNA encoding a recombinant antibody in which the CDRs and human antibody FRs are connected is constructed. The FRs of the human antibody connected via the CDRs are selected so that the CDRs form a good antigen binding site. As needed, the amino acid residues of the FRs in the antibody variable region can be appropriately modified so that the CDRs of the reconstructed human antibody form an appropriate antigen binding site (Sato, K. et al., Cancer Res. (1993) 53: 851-856). Amino acid residues in FRs to be modified include residues that directly bind to antigens via non-covalent bonds (Amit et al., Science (1986) 233: 747-53), residues that influence or act on CDR structure (Chothia et al., J. Mol. Biol. (1987) 196: 901-17), and residues involved in VH-VL interactions (EP Patent Publication No. 239400).

Humanized antibodies encoding the DNA and produced in host cells transformed or transduced with a commonly used expression vector into which the DNA is inserted can be obtained by culturing the host cells and isolating them from the culture medium.

When the antibody provided by the present invention is an antibody, a humanized antibody or a human antibody, as the C region of the antibody, it is preferred to use a C region from a human antibody. For example, as the H chain C region, Cγ1, Cγ2, Cγ3, and Cγ4 are preferably used; as the L chain C region, Cκ and Cλ are preferably used. In order to improve the stability of the antibody or its production, the human antibody C region can be appropriately modified. The chimeric antibody provided by the present invention is preferably composed of the V region of an antibody obtained from a mammal other than a human and the C region of a human antibody. On the other hand, the humanized antibody is preferably composed of the CDR of an antibody obtained from a mammal other than a human, the FR and C region of a human antibody. In addition, the human antibody is preferably composed of the CDR of an antibody obtained from a human, the FR and C region of a human antibody. The C region of a human antibody is composed of the inherent amino acid sequence corresponding to the isotypes such as IgG (IgG1, IgG2, IgG3, IgG4), IgM, IgA, IgD and IgE. As the C region of the humanized antibody provided by the present invention, it is preferred to use the C region of an antibody belonging to any isotype. The C region of human IgG is preferably used, but is not limited thereto. The FRs of human antibodies used as FRs of humanized or human antibodies are also not particularly limited, and FRs of antibodies belonging to any isotype are preferably used.

In order to reduce immunogenicity, all or part of the amino acid residues constituting the FR region can be substituted with germline sequences by using the methods described in non-patent literature (Ono K, Ohtomo T, Yoshida K, Yoshimura Y, Kawai S, Koishihara Y, Ozaki S, Kosaka M, Tsuchiya M. The humanized anti-HM1.24 antibody effectively kills multiple myeloma cells by human effector cell-mediated cytotoxicity. Mol. Immunol. (1999) 36(6): 387-395) and similar methods. Based on the reasonable prediction of low immunogenicity of germline sequences, the amino acid sequences constituting the FR regions of humanized antibodies are compared by aligning them with the amino acid sequences of the germline sequences (Abhinandan K.R. and Martin C.R., J. Mol. Biol. (2007) 369:852-862). In this comparison, the amino acid residues constituting the FR regions of different humanized antibodies can be substituted with amino acid residues in the germline sequences, within the range that does not lose antigen binding activity. Specific examples include: among the amino acid residues constituting the H chain variable region shown in SEQ ID NO: 195, the modification of L at position 70 to I, T at position 87 to R, and T at position 97 to A. In addition, among the amino acid residues constituting the L chain variable region shown in SEQ ID NO: 201, the modification of S at position 25 to A can also be mentioned.

The variable and constant regions of the modified chimeric, humanized, and human antibodies provided by the present invention may preferably be deleted, substituted, inserted, and/or added to one or more amino acids constituting the variable and constant regions of the antibodies to be modified, as long as they exhibit antigen-binding specificity.

Since chimeric antibodies, humanized antibodies, and human antibodies utilizing human-derived sequences have reduced immunogenicity in humans, they are considered useful as therapeutic antibodies to be administered to humans for therapeutic purposes.

In the methods of the present invention, known sequences can be used as gene sequences encoding the antibody H or L chain before mutation introduction. Alternatively, novel antibody gene sequences can be obtained using methods known to those skilled in the art. Such genes can be preferably obtained, for example, from an antibody library. Furthermore, such genes can be cloned using known methods such as RT-PCR using mRNA from a hybridoma producing a monoclonal antibody as a template.

Many antibody libraries are already known. Furthermore, methods for preparing antibody libraries are also known, so those skilled in the art can appropriately obtain or prepare antibody libraries. For example, preferred antibody libraries include those disclosed in Clackson et al., Nature (1991) 352:624-8; Marks et al., J. Mol. Biol. (1991) 222:581-97; Waterhouses et al., Nucleic Acids Res. (1993) 21:2265-6; Griffiths et al., EMBO J. (1994) 13:3245-60; Vaughan et al., Nature Biotechnology (1996) 14:309-14, and Japanese Patent Application Publication No. 20-504970. In addition, it is preferred to use a method for making a library in eukaryotic cells (WO95/15393 pamphlet) or a known method such as ribosome display. In addition, the technology of obtaining human antibodies by screening using a human antibody library as a starting material is also well known to those skilled in the art. That is, the variable regions of the H and L chains of the human antibody are fused in frame, and the resulting single-chain antibody (scFv) is expressed on the phage surface by phage display. By selecting a phage that binds to an antigen, the gene encoding the scFv that binds to the antigen is separated from the phage. By identifying the sequence of the gene, the DNA sequence of the variable regions of the H and L chains of the antibody that binds to the antigen can be determined. The antibody gene with the sequence is inserted into an appropriate expression vector and expressed in a suitable host cell described later, thereby appropriately obtaining human antibodies. These methods are well known, and examples thereof include the methods disclosed in WO92/01047, WO92/20791, WO93/06213, WO93/11236, WO93/19172, WO95/01438, and WO95/15388.

The method for obtaining the gene encoding the antibody from the hybridoma producing the monoclonal antibody can basically adopt the well-known technology. It is described in detail below. In short, the antibody gene is preferably obtained by the following method: according to the conventional immunization method, an animal is immunized with the desired sensitizing antigen, and then the immune cells obtained from the immunized animal are fused with the known parent cells using the conventional cell fusion method; according to the conventional screening method, the cells (hybridomas) producing the monoclonal antibody are selected, and the mRNA obtained from the selected hybridoma is used as a template to synthesize the cDNA of the antibody variable region (V region) by reverse transcriptase; and the cDNA is fused with the DNA encoding the desired antibody constant region (C region) in frame.

More specifically, preferred examples are shown below, but the present invention is not limited to these examples. The sensitizing antigen used to obtain the antibodies provided by the present invention can be a complete antigen that is immunogenic, or an incomplete antigen such as a hapten that does not exhibit immunogenicity. For example, the full-length protein or a partial polypeptide or peptide thereof can be preferably used. A preferred specific example is the soluble GPC3 core polypeptide set forth in SEQ ID NO: 207. In addition, substances composed of polysaccharides, nucleic acids, lipids, etc. are known to also function as antigens, and the antigens bound by the antibodies of the present invention are not particularly limited to the above-mentioned substance forms. The antigen is preferably prepared using methods known to those skilled in the art, for example, methods using baculovirus (e.g., WO98/46777, etc.) can be preferably used. When the immunogenicity of the antigen is low, it is preferable to immunize the animal by binding the antigen to an immunogenic macromolecule such as albumin. When the sensitizing antigen is a transmembrane molecule, a polypeptide fragment of the extracellular region of the molecule is preferably used as the sensitizing antigen, as needed. Alternatively, cells expressing the molecule on the cell surface can be preferably used as the sensitizing antigen. Furthermore, when the sensitizing antigen is an insoluble molecule, it is solubilized by binding the molecule to other water-soluble molecules, and the solubilized bound molecule is preferably used as the sensitizing antigen.

Antibody-producing cells are preferably obtained by immunizing animals with the above-mentioned appropriate sensitizing antigens. Alternatively, antibody-producing cells can be obtained by immunizing lymphocytes capable of producing antibodies in vitro. As animals to be immunized, various vertebrates and mammals can be used. In particular, rodents, lagomorphs, and primates are generally used as immunized animals. Examples include: rodents such as mice, rats, and hamsters; lagomorphs such as rabbits; and primates such as monkeys such as cynomolgus monkeys, macaques, baboons, and chimpanzees. In addition, transgenic animals that retain the full repertoire of human antibody genes in their genomes are also known, and by using such animals, human antibodies can be preferably obtained (see WO96/34096; Mendez et al., Nat. Genet. (1997) 15: 146-56). In addition to using such transgenic animals, for example, human lymphocytes can be sensitized in vitro with a desired antigen or cells expressing the desired antigen, and then fused with human myeloma cells, such as U266, to preferably obtain the desired human antibody having the antigen-binding activity (see Japanese Patent Publication No. 1-59878). Alternatively, the desired human antibody can be preferably obtained by immunizing a transgenic animal that possesses the full repertoire of human antibody genes in its genome with the desired antigen (see WO93/12227, WO92/03918, WO94/02602, WO96/34096, and WO96/33735).

Animal immunization can be carried out as follows: the sensitizing antigen is appropriately diluted and suspended with phosphate buffered saline (PBS) or physiological saline, emulsified by mixing with an adjuvant as needed, and then the sensitizing antigen is injected into the animal intraperitoneally or subcutaneously. Immunization is thus implemented. Afterwards, the sensitizing antigen mixed with Freund's incomplete adjuvant is preferably administered several times every 4 to 21 days. When confirming the production of antibodies against the sensitizing antigen in the immunized animal, the antibody titer against the sensitizing antigen in the animal serum can be measured according to conventional methods, such as enzyme-linked immunosorbent assay (ELISA), flow cytometry (FACS) and other well-known analytical methods for confirmation.

Hybridomas can be produced by fusing antibody-producing cells obtained from animals or lymphocytes immunized with the desired sensitizing antigen with myeloma cells using a fusion agent commonly used for cell fusion (e.g., polyethylene glycol) (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, 1986, 59-103). Hybridomas can be preferably produced according to, for example, the method of Milstein et al. (G. Kohler and C. Milstein, Methods Enzymol. (1981) 73: 3-46). By culturing and proliferating hybridoma cells produced by the above method, monoclonal antibodies that specifically bind to the antigen protein produced by the hybridoma are obtained. The binding specificity of the monoclonal antibody to the antigen protein can be appropriately measured by known analytical methods such as immunoprecipitation, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), and flow cytometry (FACS). Thereafter, if necessary, the hybridoma producing the antibody whose desired specificity, binding activity, or activity has been determined can be appropriately subcloned by a method such as limiting dilution to isolate the monoclonal antibody produced by the hybridoma.

Next, regarding the gene encoding the selected antibody, the gene can be cloned from the above-mentioned hybridoma or antibody-producing cells (sensitized lymphocytes, etc.) using a probe that can specifically bind to the gene (e.g., an oligonucleotide complementary to the sequence encoding the antibody constant region). In addition, the gene can also be cloned by RT-PCR using mRNA obtained from the hybridoma or antibody-producing cells (sensitized lymphocytes, etc.) as a template. Immunoglobulins are divided into five different classes based on their structure and function: IgA, IgD, IgE, IgG, and IgM. Each class is further divided into several isotypes (e.g., IgG1, IgG2, IgG3, and IgG4; IgA1 and IgA2, etc.). The antibodies provided by the present invention can be derived from antibodies belonging to any class and subclass therein, and there is no particular limitation to any class and subclass, but antibodies belonging to the IgG class are particularly preferred.

The genes encoding the amino acid sequences constituting the H and L chains of antibodies can be appropriately modified using genetic engineering methods. For example, by modifying the nucleic acid residues encoding the amino acid sequences of antibodies such as mouse antibodies, rat antibodies, rabbit antibodies, hamster antibodies, sheep antibodies, and camel antibodies, gene recombinant antibodies, such as chimeric antibodies and humanized antibodies, can be appropriately prepared. The gene recombinant antibodies have been artificially modified to reduce their heterologous antigenicity to humans. Chimeric antibodies are antibodies composed of the H and L chain variable regions of antibodies from mammals other than humans, such as mice, and the H and L chain constant regions of human antibodies. For example, DNA encoding the variable regions of antibodies from mice is connected to DNA encoding the constant regions of human antibodies, which are integrated into expression vectors. The resulting recombinant vectors are introduced into hosts for expression, thereby obtaining chimeric antibodies. Humanized antibodies, also known as reshaped human antibodies, are antibodies in which the complementary determining regions (CDRs) of antibodies isolated from mammals other than humans, such as mice, are connected in frame with the framework regions of human antibodies to form a codon sequence. The DNA sequence encoding the humanized antibody can be synthesized by overlapping PCR using multiple oligonucleotides as templates. The materials and methods for overlapping PCR are described in WO98/13388 and the like.

The DNA encoding the variable region of the genetically recombinant antibody of the present invention can be prepared by making multiple oligonucleotides having overlapping nucleotide sequences and obtaining them by overlapping PCR reaction. The oligonucleotides are then ligated in frame with the DNA encoding the constant region of the human antibody to form a codon sequence. The DNA ligated in this manner is then inserted into an expression vector to express the DNA, and the vector is then introduced into a host. The host is cultured to express the antibody encoded by the DNA. The expressed antibody can be obtained by appropriately purifying the host culture medium (see EP239400; WO96/02576). The FRs of the humanized antibody connected via the CDRs are selected so that the complementarity determining regions form a good antigen binding site with the antigen. If necessary, the amino acid residues constituting the selected antibody variable region can be appropriately substituted and modified so that the complementarity determining regions of the reconstructed human antibody form a suitable antigen binding site with the antigen (K. Sato et al., Cancer Res. (1993) 53, 851-856).

In addition to the modifications involved in the above-mentioned humanization, modifications can be implemented to improve the biological properties of antibodies such as the binding activity of antibodies to the antigens they recognize. The modifications in the present invention can preferably be performed by methods such as site-specific mutations (for example, with reference to Kunkel, Proc. Natl. Acad. Sci. USA (1985) 82, 488), PCR mutagenesis, and cassette mutagenesis. Generally, the amino acid sequence constituting the modified antibody with improved biological properties has a homology and/or similarity of 70% or more, more preferably 80% or more, and further preferably 90% or more (for example, 95% or more, 97%, 98%, 99%, etc.) to the amino acid sequence constituting the antibody to be modified (i.e., the original antibody as the modified antibody). In this specification, the homology and/or similarity of the sequence refers to the ratio of amino acid residues that are identical (identical residues) or similar (amino acid residues classified into the same group according to the side chain characteristics of common amino acids) to the amino acid residues constituting the original antibody as the modified antibody after the sequence is aligned and gaps are introduced as needed to maximize the sequence homology. In general, natural amino acid residues can be divided into the following groups based on the properties of their side chains: (1) hydrophobic: alanine, isoleucine, valine, methionine, and leucine; (2) neutral hydrophilic: asparagine, glutamine, cysteine, threonine, and serine; (3) acidic: aspartic acid and glutamic acid; (4) basic: arginine, histidine, and lysine; (5) residues that affect chain orientation: glycine and proline; and (6) aromatic: tyrosine, tryptophan, and phenylalanine.

As a modification for the purpose of enhancing antibody function, a specific method is, for example, to improve the cytotoxicity exerted by antibodies represented by humanized antibodies. As cytotoxicity, preferred examples include: antibody-dependent cell-mediated cytotoxicity (ADCC) activity, complement-dependent cytotoxicity (CDC) activity, etc. In the present invention, CDC activity refers to the cytotoxicity produced by the complement system. ADCC activity refers to the activity of cells (immune cells, etc.) with Fcγ receptors binding to their Fc parts via Fcγ receptors when specific antibodies are attached to cell surface antigens of target cells, causing damage to target cells. Whether the test antibody has ADCC activity or CDC activity can be determined according to known methods (for example, Current protocols in Immunology, Chapter 7. Immunologic studies in humans, Editor, John E, Coligan et al., John Wiley & Sons, Inc., (1993) etc.).

Specifically, effector cells, complement solution, and target cells are first prepared.

(1) Preparation of effector cells

Spleens are removed from CBA/N mice, etc., and spleen cells are isolated in RPMI1640 medium (Invitrogen). After washing with the same medium supplemented with 10% fetal bovine serum (FBS, HyClone), the cell concentration is adjusted to 5×10 ⁶ cells/mL to prepare effector cells.

(2) Preparation of complement solution

Baby Rabbit Complement (CEDARLANE) was diluted 10-fold with a culture medium (Invitrogen) containing 10% FBS to prepare a complement solution.

(3) Preparation of target cells

The cells expressing the antigen protein to which the test antibody binds are cultured in DMEM medium containing 0.2mCi 51Cr-sodium chromate (GE Healthcare Bioscience) and 10% FBS at 37°C for 1 hour, thereby radiolabeling the target cells. As cells expressing the antigen protein to which the test antibody binds, cells transformed with genes encoding the antigen protein to which the test antibody binds, ovarian cancer, prostate cancer, breast cancer, uterine cancer, liver cancer, lung cancer, pancreatic cancer, gastric cancer, bladder cancer, and colorectal cancer cells can be used. After radiolabeling, the cells are washed three times with RPMI1640 medium containing 10% FBS, and the cell concentration is adjusted to 2×10 ⁵ cells/mL to prepare the target cells.

ADCC activity or CDC activity can be measured according to the following method. When measuring ADCC activity, 50 μL of target cells and 50 μL of test antibody are added to a 96-well round-bottom plate (Becton Dickinson) and reacted on ice for 15 minutes. Afterwards, the reaction mixture to which 100 μL of effector cells are added is cultured in a _CO2 incubator for 4 hours. The final concentration of the test antibody can be appropriately used in the range of 0 to 10 μg/mL. After incubation, 100 μL of supernatant is recovered and the radioactivity of the supernatant is measured using a γ counter (COBRAII AUTO-GAMMA, MODEL D5005, Packard Instrument Company). Cytotoxicity (%) can be calculated using the obtained radioactivity value according to the formula (AC)/(BC)×100. A shows the radioactivity (cpm) when using each test antibody sample, B shows the radioactivity (cpm) when using a sample supplemented with 1% NP-40 (nacalai tesque), and C shows the radioactivity (cpm) when using a sample containing only target cells.

On the other hand, when measuring CDC activity, 50 μL of target cells and 50 μL of test antibody were added to a 96-well flat-bottom plate (Becton Dickinson) and reacted on ice for 15 minutes. Afterwards, the reaction mixture to which 100 μL of complement solution was added was cultured in a CO ₂ incubator for 4 hours. The final concentration of the test antibody can be appropriately used within the range of 0 to 3 μg/mL. After incubation, 100 μL of supernatant was recovered and the radioactivity of the supernatant was measured using a γ counter. Cytotoxicity can be calculated by performing the same operation as for the determination of ADCC activity.

When measuring the cytotoxicity produced by the antibody conjugate, 50 μL of target cells and 50 μL of the test antibody conjugate were added to a 96-well flat-bottom plate (Becton Dickinson) and reacted on ice for 15 minutes. The plate was incubated in a _CO2 incubator for 1 to 4 hours. The final concentration of the antibody can be appropriately used in the range of 0 to 3 μg/mL. After incubation, 100 μL of the supernatant was recovered and the radioactivity of the supernatant was measured using a gamma counter. Cytotoxicity can be calculated by performing the same operation as for the determination of ADCC activity.

As described above, the variable regions of antibody H and L chains are generally composed of three CDRs and four FRs. In a preferred embodiment of the present invention, the amino acid residues to be "modified" can be appropriately selected from, for example, the amino acid residues constituting the CDRs or FRs.

Those skilled in the art can preferably obtain the amino acid sequences of FRs constituting antibody variable regions, that is, sequences actually existing in organisms such as humans and mice, using public databases such as Kabat.

In a preferred embodiment of the present invention, a humanized antibody whose plasma pharmacokinetics are controlled by the method of the present invention is provided. This humanized antibody, for example, comprises a complementarity determining region (CDR) derived from a non-human animal, a framework region (FR) derived from a human, and a human C region, wherein at least one amino acid residue in the CDR or FR that can be exposed on the antibody surface has an amino acid residue with a different charge from the amino acid residue at the position corresponding to the CDR or FR of the original antibody, and the plasma pharmacokinetics of the humanized antibody are controlled compared to a chimeric antibody having the same C region.

Furthermore, in a preferred embodiment of the present invention, a human antibody whose plasma pharmacokinetics are controlled by the method of the present invention is provided. This human antibody, for example, comprises a complementarity determining region (CDR) derived from a human, a framework region (FR) derived from a human, and a human C region, wherein at least one amino acid residue in the CDR or FR that can be exposed on the antibody surface has an amino acid residue with a different charge from the amino acid residue at the position corresponding to the CDR or FR of the original antibody, and the plasma pharmacokinetics of the human antibody are controlled compared to a chimeric antibody having the same C region.

The human constant region preferably comprises a wild-type human Fc region, but modified Fc regions are also preferably used. This "modified Fc" encompasses Fc regions in which the amino acid residues constituting the Fc region have been modified, as well as Fc regions in which the modifications to the Fc portion have been altered. A preferred example of such alterations is altering the pattern of sugar chain modifications added to the Fc portion. In this specification, a preferred example is the "antibody with reduced fucose content that binds to the Fc region of an antibody" specifically disclosed as a reference example.

An "antibody with reduced fucose content bound to the antibody Fc region" refers to an antibody that has significantly less fucose bound, preferably no detectable fucose, compared to a control antibody. Fucose is typically attached to N-glycosidically bonded sugar chains bound to two glycosylation sites in the Fc region of two H chains that constitute a single antibody molecule. In the present invention, an "antibody with reduced fucose content bound to the antibody Fc region" refers to an antibody that, when compared to such a conventional antibody as a control, has a fucose content that is 50% or less, preferably 25% or less, more preferably 10% or less, and particularly preferably 0% or less of the total sugar chain content of the control antibody. Fucose content can be measured using the analytical methods specifically exemplified in the Reference Examples below. In addition to the methods described in the reference examples of the present invention, preferred examples of methods for producing antibodies with reduced fucose content include methods using animal cells lacking fucosyltransferase (Biotechnol Bioeng. (2004), 87(5), 614-22), methods using animal cells modified with complex branched sugar chains (Biotechnol Bioeng. (2006) 93(5), 851-61), etc. Preferred examples of methods for producing antibodies using cells other than animal cells as host cells include methods using plant cells (Nature Biotechnology (2006) 24, 1591-7), and methods using yeast cells (Nature Biotechnology (2006) 24, 210-5).

A preferred embodiment of the production method of the present invention is a method for producing a polypeptide comprising an antibody variable region with a modified isoelectric point, the method comprising:

(a) modifying a nucleic acid encoding a polypeptide comprising amino acid residues to change the charge of at least one of the amino acid residues that can be exposed on the surface of the CDR region of the polypeptide;

(b) culturing the host cell to express the nucleic acid;

(c) recovering the polypeptide comprising the antibody variable region from the host cell culture.

A preferred embodiment of the production method of the present invention is a method for producing a polypeptide comprising an antibody variable region with controlled pharmacokinetics in plasma, the method comprising:

(a) modifying a nucleic acid encoding a polypeptide comprising amino acid residues to change the charge of at least one of the amino acid residues that can be exposed on the surface of the CDR region of the polypeptide;

(b) culturing the host cell to express the nucleic acid;

(c) recovering the polypeptide comprising the antibody variable region from the host cell culture.

Furthermore, polypeptides comprising antibody variable regions and having controlled plasma pharmacokinetics produced by this method are also encompassed by the present invention.

The present invention also provides a method for producing a multispecific polypeptide, wherein the multispecific polypeptide comprises a first polypeptide and a second polypeptide having an antibody variable region. The present invention further provides a multispecific polypeptide produced according to the method. As a preferred embodiment of the production method of the present invention, the method comprises modifying a nucleic acid encoding the amino acid residues of the first polypeptide and/or a nucleic acid encoding the amino acid residues of the second polypeptide so as to increase the difference in isoelectric point between the first polypeptide and the second polypeptide. That is, by changing the charge of the amino acid residues of the first polypeptide and the second polypeptide, the difference in the isoelectric point (pI) of the polypeptides is increased, and a multispecific antibody can be produced by utilizing the difference in the isoelectric point. In detail, the production method comprises the following steps (a) to (c).

(a) comprising modifying a nucleic acid encoding a polypeptide comprising an amino acid residue to change the charge of at least one of the amino acid residues that can be exposed on the surface of the CDR region of a first polypeptide and a second polypeptide, wherein the modification of the nucleic acid refers to modifying the nucleic acid encoding the amino acid residue of the first polypeptide and/or the nucleic acid encoding the amino acid residue of the second polypeptide so that the difference between the isoelectric points of the first polypeptide and the second polypeptide is increased compared to before the modification;

(b) culturing the host cell to express the nucleic acid;

(c) Recovering the multispecific antibody from the host cell culture.

The polypeptides herein generally refer to peptides and proteins having a length of approximately 10 amino acids or more. These are typically polypeptides derived from organisms, but are not particularly limited and may include, for example, polypeptides containing artificial sequences. They may also be natural polypeptides, synthetic polypeptides, recombinant polypeptides, or any other polypeptide. Furthermore, fragments of the aforementioned polypeptides are also encompassed by the polypeptides of the present invention.

In the present invention, a "multispecific polypeptide comprising a first polypeptide and a second polypeptide having an antibody variable region" refers to a polypeptide comprising an antibody variable region that binds to at least two or more different antigens or different epitopes within the same antigen. As described above, polypeptides comprising an antibody variable region include, for example, antibodies, small molecule antibodies, scaffold proteins, etc.

In the present invention, "increasing the difference in isoelectric points of polypeptides" means that the isoelectric points of two or more polypeptides are made unequal, or the difference in isoelectric points between the two or more polypeptides is increased, by altering the surface amino acid charges. The difference in isoelectric points can be observed, for example, using methods such as isoelectric electrophoresis. In the present invention, it is preferred to control the isoelectric point while maintaining the structure and function (activity) of the polypeptide.

That is, the present invention provides a method for producing a multispecific antibody, wherein the multispecific polypeptide comprises a first polypeptide having an antibody variable region and a second polypeptide, the method comprising:

(a) modifying the nucleic acid encoding the amino acid residues of the first polypeptide and/or the nucleic acid encoding the amino acid residues of the second polypeptide to change the charge of at least one amino acid residue that can be exposed on the surface of the CDR region, so that the difference in isoelectric point between the first polypeptide and the second polypeptide is 1.0 or more, preferably 1.2 or more, and more preferably 1.5 or more;

(b) culturing the host cell to express the nucleic acid;

(c) Recovering the multispecific antibody from the host cell culture.

The present invention also provides a method for modifying a multispecific polypeptide, which modification method is used to purify a multispecific polypeptide comprising a first polypeptide and a second polypeptide having an antibody variable region. As a preferred embodiment of the purification method of the present invention, it includes modifying a nucleic acid encoding the amino acid residues of the first polypeptide and/or a nucleic acid encoding the amino acid residues of the second polypeptide to increase the difference in isoelectric point between the first polypeptide and the second polypeptide. That is, by changing the charge of the amino acid residues of the first polypeptide and the second polypeptide, a difference in isoelectric point (pI) is introduced into the polypeptides, and the difference in isoelectric point can be utilized to purify the multispecific antibody. In detail, the purification method includes the following steps (a) to (c).

(a) comprising modifying a nucleic acid encoding a polypeptide comprising an amino acid residue to change the charge of at least one of the amino acid residues that can be exposed on the surface of the CDR region of a first polypeptide and a second polypeptide, wherein the modification of the nucleic acid refers to modifying the nucleic acid encoding the amino acid residue of the first polypeptide and/or the nucleic acid encoding the amino acid residue of the second polypeptide so that the difference between the isoelectric points of the first polypeptide and the second polypeptide is increased compared to before the modification;

(b) culturing the host cell to express the nucleic acid;

(c) Purifying the multispecific antibody from the host cell culture using standard chromatography methods.

It should be noted that a method for producing a multispecific antibody including a purification step using the above-mentioned purification method is also included in the present invention.

In the above-mentioned method of the present invention, "modified nucleic acid" refers to a modified nucleic acid sequence so as to form a codon corresponding to the amino acid residue introduced by the "modification" in the present invention. More specifically, "modified nucleic acid" refers to a nucleic acid that modifies the codon constituting the modified codon so as to make the codon equivalent to the amino acid residue before modification become the codon for the amino acid residue introduced by modification. It generally refers to a genetic manipulation or mutation treatment such as replacing at least one base of the nucleic acid constituting the codon so as to become a codon encoding the target amino acid residue. That is, the codon encoding the amino acid residue for modification is replaced by the codon encoding the amino acid residue introduced by modification. Regarding such nucleic acid modification, those skilled in the art can adopt known techniques, such as site-specific mutagenesis, PCR mutation introduction method, etc. to appropriately carry out.

Typically, the nucleic acid of the present invention is cloned (inserted) into an appropriate vector and introduced into a host cell. The vector is not particularly limited, as long as the inserted nucleic acid is stably maintained. For example, when using Escherichia coli as a host, pBluescript vectors (Stratagene) are preferred as cloning vectors, but various commercially available vectors can also be used. When using vectors to produce the polypeptides of the present invention, expression vectors are particularly practical. There are no particular limitations on the expression vector, as long as it is a vector that expresses the polypeptide in a test tube, in Escherichia coli, in cultured cells, or in an organism. For example, when expressing the polypeptide in a test tube, the pBEST vector (Promega) is preferred; when expressing the polypeptide in Escherichia coli, the pET vector (Invitrogen) is preferred; when expressing the polypeptide in cultured cells, the pME18S-FL3 vector (GenBank Accession No. AB009864) is preferred; when expressing the polypeptide in an organism, the pME18S vector (Mol Cell Biol. (1988) 8, 466-472) is preferred. Insertion of the DNA of the present invention into the vector can be preferably performed using conventional methods, for example, ligase reaction using restriction enzyme sites (Current protocols in Molecular Biology edited by Ausubel et al. (1987) published by John Wiley & Sons. Section 11.4-11.11).

The host cells are not particularly limited, and various host cells can be used depending on the intended purpose. Examples of cells for expressing polypeptides include bacterial cells (e.g., Streptococcus, Staphylococcus, Escherichia coli, Streptomyces, and Bacillus subtilis), fungal cells (e.g., yeast and Aspergillus), insect cells (e.g., Drosophila S2 and Spodoptera SF9), animal cells (e.g., CHO, COS, HeLa, C127, 3T3, BHK, HEK293, and Bowes melanoma cells), and plant cells. The vector can be introduced into the host cells by known methods such as calcium phosphate precipitation, electroporation (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley & Sons. Section 9.1-9.9), lipofection, and microinjection.

It is preferred to incorporate an appropriate secretion signal into the antibody of interest so that the polypeptide (antibody) expressed in the host cell is secreted into the lumen of the endoplasmic reticulum, the periplasmic space, or the extracellular environment. The above-mentioned signal can preferably be an endogenous signal or a heterologous signal specific to the polypeptide (antibody) of interest.

In the above production method, the polypeptide (antibody) is recovered by recovering the culture medium if the polypeptide (antibody) of the present invention is secreted into the culture medium. If the antibody of the present invention is produced intracellularly, the cells are first lysed and then the antibody is recovered.

To purify the antibodies of the present invention recovered from recombinant cell culture, the following well-known methods can be preferably used: ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography, and lectin chromatography.

In the present invention, the polypeptides that modify nucleic acids are preferably homomultimers of the first polypeptide, homomultimers of the second polypeptide, and heteromultimers of the first and second polypeptides. Examples of the homomultimers of the first polypeptide, the homomultimers of the second polypeptide, and the heteromultimers of the first and second polypeptides include, but are not limited to, those described in the Examples.

Standard chromatography methods used in the present invention include, but are not limited to, cation exchange chromatography, anion exchange chromatography, hydrophobic chromatography, hydroxyapatite chromatography, hydrophobic charge interaction chromatography, and chromatofocusing.

In the above method of the present invention, the first polypeptide and the second polypeptide preferably comprise a heavy chain variable region (VH). The variable region may comprise, for example, a complementarity determining region (CDR) and a framework region (FR).

Furthermore, in the above-mentioned method of the present invention, the variable region of the multispecific polypeptide preferably comprises a light chain variable region.

Furthermore, in the above-described method of the present invention, the first and second polypeptides preferably comprise heavy chain constant regions. More preferably, the heavy chain constant regions produce a difference in pi between the first and second polypeptides. Such heavy chain constant regions include those of antibodies with a difference in pi. The pi difference can be introduced into the first and second polypeptides using the heavy chain constant regions of IgG1, IgG2, IgG3, or IgG4, which already have a difference in pi. Alternatively, non-wild-type human constant regions can be created by modifying only the amino acids in the heavy chain constant regions of the first and second polypeptides that result in a difference in isoelectric point between these subclasses, or by simultaneously modifying adjacent amino acids that do not affect their isoelectric points, thereby introducing a difference in pi between the two constant regions. Examples of modification sites for introducing a pI difference into the constant region include positions 137, 196, 203, 214, 217, 233, 268, 274, 276, 297, 355, 392, 419, and 435 in the H chain constant region according to EU numbering.

In addition, since the pI difference is caused by removing the sugar chain in the heavy chain constant region, the modification site for introducing the pI chain is also the 297th glycosylation site.

In the present invention, the method in which the first polypeptide and the second polypeptide contain a heavy chain constant region also includes a method in which the first polypeptide and the second polypeptide contain a heavy chain variable region and/or a method in which the multispecific antibody contains a third polypeptide containing a light chain variable region, and the first polypeptide and the second polypeptide respectively form a multimer with the third polypeptide.

Furthermore, multispecific polypeptides produced by the above-mentioned methods are also included in the present invention.

Furthermore, when the first polypeptide in the multispecific antibody provided by the present invention comprises a heavy chain variable region, the aforementioned "increase in isoelectric point difference" can be achieved, for example, by providing a charge to at least one amino acid residue selected from the group consisting of amino acid residues at positions 31, 61, 62, 64, and 65 according to Kabat numbering in the heavy chain variable region. When the first polypeptide comprises a light chain variable region, the aforementioned "increase in isoelectric point difference" can be achieved, for example, by providing a charge to at least one amino acid residue selected from the group consisting of amino acid residues at positions 24, 27, 53, 54, and 55 according to Kabat numbering in the light chain variable region. Among the amino acid residues in the first polypeptide numbered above, amino acid residues other than the charged amino acid residues may have the same charge as, be uncharged, or have an opposite charge as the charged amino acid residue, as long as they contribute to a difference in isoelectric point between the first and second polypeptides.

The multispecific antibody of the present invention is characterized in that the second polypeptide preferably has a charge opposite to that of the charged amino acid residues of the first polypeptide, or is uncharged. Specifically, the multispecific antibody is a multispecific antibody in which the second polypeptide comprises a heavy chain variable region, and at least one amino acid residue selected from amino acid residues at positions 31, 61, 62, 64, and 65 according to Kabat numbering in the heavy chain variable region has a charge opposite to that of the charged amino acid residues selected in the variable region of the first polypeptide, or is uncharged. When the multispecific antibody comprises a light chain variable region, the multispecific antibody is a multispecific antibody in which at least one amino acid residue selected from amino acid residues at positions 24, 27, 53, 54, and 55 according to Kabat numbering in the light chain variable region has a charge opposite to that of the charged amino acid residues selected in the variable region of the first polypeptide, or is uncharged. Among the amino acid residues of the second polypeptide indicated by the above numbering, the amino acid residues other than the charged amino acid residues may have the same charge as the charged amino acid residue, may be uncharged, or may have an opposite charge, as long as they cause a difference in the isoelectric point between the first polypeptide and the second polypeptide.

Furthermore, when the multispecific antibody comprises an antibody constant region, in order to lower its isoelectric point, for example, preferably the following sequence is used at position 137: an IgG2 or IgG4 sequence; at position 196: an IgG1, IgG2, or IgG4 sequence; at position 203: an IgG2 or IgG4 sequence; at position 214: an IgG2 sequence; at position 217: an IgG1, IgG3, or IgG4 sequence; at position 233: an IgG1, IgG3, or IgG4 sequence; at position 268: an IgG4 sequence; at position 274: an IgG2, IgG3, or IgG4 sequence; at position 276: an IgG1, IgG2, or IgG4 sequence; at position 355: an IgG4 sequence; at position 392: an IgG3 sequence; at position 419: an IgG4 sequence; and at position 435: an IgG1, IgG2, or IgG4 sequence. In order to increase the isoelectric point, for example, preferably, the sequence of IgG1 or IgG3 is used at position 137, the sequence of IgG3 is used at position 196, the sequence of IgG1 or IgG3 is used at position 203, the sequence of IgG1, IgG3 or IgG4 is used at position 214, the sequence of IgG2 is used at position 217, the sequence of IgG2 is used at position 233, the sequence of IgG2 is used at position 268, the sequence of IgG1, IgG2 or IgG3 is used at position 274, the sequence of IgG1 is used at position 276, the sequence of IgG3 is used at position 355, the sequence of IgG1, IgG2 or IgG3 is used at position 392, the sequence of IgG1, IgG2 or IgG4 is used at position 419, the sequence of IgG1, IgG2 or IgG3 is used, and the sequence of IgG3 is used at position 435.

These sequences can be used as long as they provide a sufficient difference in isoelectric points between the two H chains, and not all sequences are necessarily used.

In the above antibodies, "having the same charge" means, for example, that the amino acid residues in the heavy chain variable region according to the above Kabat numbering or the amino acid residues in the heavy chain constant region according to the above EU numbering have amino acid residues included in either group (a) or (b) below.

(a) Glutamic acid (E), aspartic acid (D)

(b) Lysine (K), Arginine (R), Histidine (H)

"Having opposite charges" means, for example, that in the second polypeptide having a heavy chain variable region and/or a heavy chain constant region, at least one of the amino acid residues having the above-mentioned Kabat numbering or the above-mentioned EU numbering is the amino acid residue at the corresponding position in the heavy chain variable region and/or heavy chain constant region contained in the first polypeptide, and when the second polypeptide has amino acid residues contained in either group (a) or (b) above, the remaining amino acid residues are amino acid residues contained in a different group.

That is, the present invention provides a multispecific antibody, wherein the amino acid residues having the same charge are selected from the amino acid residues contained in any one of the groups (a) or (b) above.

It should be noted that when the original (before modification) amino acid residue is already charged, it is modified into an uncharged amino acid residue, which is also one of the preferred embodiments of the present invention.

In the present invention, it is preferred to modify the amino acid residues so as to increase the difference in isoelectric point (pI) between the first polypeptide and the second polypeptide. When there are multiple amino acid residues introduced by modification, these amino acid residues may include a small number of uncharged amino acid residues.

The present invention also relates to a composition (medicament) comprising a polypeptide (eg, an antibody such as IgG antibody) whose plasma pharmacokinetics are controlled by the method of the present invention and a pharmaceutically acceptable carrier.

In the present invention, a pharmaceutical composition generally refers to a drug used for the treatment or prevention, or examination and diagnosis of a disease.

The pharmaceutical composition of the present invention can be preferably prepared according to methods well known to those skilled in the art. For example, the pharmaceutical composition can be prepared into the form of injections such as sterile solutions or suspensions with water or a pharmaceutically acceptable liquid other than water for parenteral administration. For example, it is preferably combined with a pharmacologically acceptable carrier or medium, specifically sterile water or physiological saline, vegetable oil, emulsifier, suspending agent, surfactant, stabilizer, flavoring agent, excipient, vehicle, preservative, adhesive, etc., and mixed according to the unit dosage form required by the pharmaceutical rules (pharmaceutical manufacturing) of conventional approval to prepare the preparation. The amount of active ingredient in these preparations is set to the appropriate dosage to obtain the indicated range.

The sterile composition for injection can be formulated preferably using a vehicle such as distilled water for injection according to conventional pharmaceutical preparation regulations.

Examples of aqueous solutions for injection include physiological saline and isotonic solutions containing glucose or other adjuvants (e.g., D-sorbitol, D-mannose, D-mannitol, sodium chloride). These aqueous solutions for injection may be used in combination with appropriate cosolvents, such as alcohols (e.g., ethanol), polyols (e.g., propylene glycol, polyethylene glycol), and nonionic surfactants (e.g., polysorbate 80™, HCO-50).

Oily liquids include sesame oil and soybean oil. These oily liquids can be appropriately combined with benzyl benzoate and/or benzyl alcohol as cosolvents. They can also be appropriately mixed with buffers (e.g., phosphate buffer and sodium acetate buffer), analgesics (e.g., procaine hydrochloride), stabilizers (e.g., benzyl alcohol and phenol), and antioxidants. The injection prepared in this manner is typically filled into a suitable ampoule.

The pharmaceutical composition of the present invention can preferably be administered parenterally. For example, it can be suitably prepared in the form of a composition for injection, nasal administration, transpulmonary administration, or transdermal administration. For example, it can be appropriately administered systemically or locally by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, or the like.

The method of administration can be appropriately selected according to the age and symptoms of the patient. The dosage of the pharmaceutical composition containing the antibody or the polynucleotide encoding the antibody can be appropriately set to, for example, the range of 0.0001 mg to 1000 mg/kg body weight per time. Alternatively, for example, a dosage of 0.001 to 100,000 mg per patient can be set or prepared, but the present invention is not limited to the above numerical values. The dosage and method of administration can vary according to the patient's weight, age, symptoms, etc., but those skilled in the art can consider these conditions and set appropriate dosage and method of administration.

The present invention also provides nucleic acids encoding antibodies (eg, humanized glypican 3 antibodies) whose plasma pharmacokinetics are controlled according to the methods of the present invention. Furthermore, vectors carrying the nucleic acids are also encompassed by the present invention.

The present invention also provides a host cell containing the above-mentioned nucleic acid. The type of the host cell is not particularly limited, and examples include bacterial cells such as Escherichia coli and various animal cells. The host cell can be preferably used as a production system for producing and expressing the antibody of the present invention. That is, the present invention provides a production system for producing antibodies using the host cell. As the production system, in vitro and in vivo production systems can be preferably used. As host cells used in in vitro production systems, eukaryotic cells and prokaryotic cells are preferably used.

Examples of eukaryotic cells used as host cells include animal cells, plant cells, and fungal cells. Preferred examples of animal cells include mammalian cells such as CHO (J. Exp. Med. (1995) 108, 945), COS, HEK293, 3T3, myeloma cells, BHK (baby hamster kidney), HeLa, and Vero cells; amphibian cells such as Xenopus laevis oocytes (Valle et al., Nature (1981) 291: 338-340); and insect cells such as Sf9, Sf21, and Tn5. For expression of the antibodies of the present invention, CHO-DG44, CHO-DX11B, COS7 cells, HEK293 cells, and BHK cells are preferably used. For large-scale expression using animal cells, CHO cells are particularly preferred as host cells. The introduction of recombinant vectors into host cells is preferably carried out by, for example, the calcium phosphate method, the DEAE-dextran method, a method using cationic liposome DOTAP (Boehringer Mannheim), electroporation, lipofection, or the like.

Plant cells include, for example, cells derived from tobacco and duckweed (Lemna minor), which are known as protein production systems, and the antibodies of the present invention can be produced by callus culture of these cells. Protein expression systems using fungal cells are well known, and these fungal cells can be used as host cells for producing the antibodies of the present invention. Examples of such fungal cells include yeast, such as cells of the genus Saccharomyces (Saccharomyces cerevisiae, Schizosaccharomyces pombe, etc.), and filamentous fungi, such as cells of the genus Aspergillus (Aspergillus niger, etc.).

When using prokaryotic cells, a production system utilizing bacterial cells is preferably used. In addition to the aforementioned Escherichia coli (E. coli), a production system utilizing Bacillus subtilis (B. subtilis) is also known as a bacterial cell, and these bacterial cells can be preferably used to produce the antibodies of the present invention.

In order to produce antibodies using the host cells of the present invention, host cells transformed with an expression vector containing a polynucleotide encoding the antibody of the present invention are cultured. The polynucleotide encoding the antibody is expressed in this culture. The culture is preferably carried out according to a known method. For example, when animal cells are used as hosts, DMEM, MEM, RPMI1640, or IMDM can be preferably used as the culture medium. In this case, a serum supplement such as FBS or fetal calf serum (FCS) is preferably used in combination. Cells can also be cultured in a serum-free culture. Although the culture conditions depend on the host cells, the culture can preferably be carried out at a pH of about 6 to 8. The culture is usually carried out at about 30 to 40°C for about 15 to 200 hours, and the culture medium can be exchanged or aerated and stirred as needed.

On the other hand, as systems for producing the antibodies of the present invention in vivo, for example, production systems using animals or plants can be preferably used. That is, a polynucleotide encoding the antibodies of the present invention is introduced into these animals or plants, and the glypican 3 antibodies are produced in the animals or plants and recovered. The "host" of the present invention includes the above-mentioned animals and plants.

When using animals as hosts, production systems using mammals or insects can be utilized. As mammals, goats, pigs, sheep, mice, cattle, etc. are preferably used (Vicki Glaser, SPECTRUM Biotechnology Applications (1993)). When using mammals, transgenic animals can be used.

For example, a polynucleotide encoding an antibody of the present invention can be made into a fusion gene with a gene encoding a polypeptide specifically produced in milk (e.g., goat β-casein). Then, the polynucleotide fragment containing the fusion gene is injected into a goat embryo, and the embryo is transplanted into a female goat. The goat that received the embryo gives birth to a transgenic goat, and the target antibody can be obtained from the milk produced by the transgenic goat or its offspring. In order to increase the amount of milk containing the antibody produced by the transgenic goat, it is preferable to administer appropriate hormones to the transgenic goat (Ebert et al., Bio/Technology (1994) 12, 699-702).

Insects that produce the antibodies of the present invention can be silkworms, for example. When silkworms are used, the silkworms are infected with a baculovirus having a polynucleotide encoding the target antibody inserted into the viral genome, and the target glypican 3 antibody is obtained from the body fluids of the infected silkworms (Susumu et al., Nature (1985) 315, 592-4).

Furthermore, when plants are used to produce the antibodies of the present invention, tobacco plants can be used, for example. When using tobacco, a polynucleotide encoding the desired antibody is inserted into a plant expression vector, such as pMON 530, and the resulting recombinant vector is then introduced into bacteria such as Agrobacterium tumefaciens. Tobacco, such as Nicotiana tabacum, is infected with this bacterium (Ma et al., Eur. J. Immunol. (1994) 24, 131-8), and the desired glypican 3 antibody is obtained from the infected tobacco leaves. Duckweed is infected with the same bacteria, and the desired glypican 3 antibody is obtained from the infected duckweed cells that have formed colonies (Cox KM et al., Nat. Biotechnol. (2006) 24(12), 1591-7).

The antibodies of the present invention thus obtained can be separated from the host cell or extracellularly (culture medium, milk, etc.) and purified into substantially pure and uniform antibodies. The separation and purification of antibodies can preferably use the separation and purification methods commonly used in the purification of polypeptides, but are not limited to these methods. For example, chromatography columns, filters, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric point electrophoresis, dialysis, recrystallization, etc. can be appropriately selected or combined to separate and purify antibodies.

Examples of chromatography include affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration chromatography, reverse phase chromatography, and adsorption chromatography (Strategies for Protein Purification and Characterization: A Laboratory Course Manual. Ed. Daniel R. Marshark et al. (1996) Cold Spring Harbor Laboratory Press). These chromatography methods can be performed using liquid chromatography, such as HPLC and FPLC. Examples of columns used for affinity chromatography include Protein A columns and Protein G columns. Examples of columns using Protein A include HyperD, POROS, and Sepharose F.F. (Pharmacia).

As described above, a method for producing an antibody of the present invention having controlled pharmacokinetics in plasma is also one of the preferred embodiments of the present invention. The method comprises the steps of culturing the host cell of the present invention and recovering the anti-glypican 3 antibody from the cell culture.

The present invention provides pharmaceutical compositions comprising second-generation molecules superior to tocilizumab, and methods for producing such pharmaceutical compositions. These second-generation molecules superior to tocilizumab are derived by modifying the amino acid sequences of the variable and constant regions of tocilizumab, a humanized anti-IL-6 receptor IgG1 antibody. This enhances efficacy and improves plasma retention, thereby reducing dosing frequency and maintaining sustained therapeutic effects. Furthermore, the molecules improve immunogenicity, safety, and physicochemical properties, and methods for producing such pharmaceutical compositions are also provided. The present invention also provides antibody constant regions suitable for use as pharmaceuticals.

The present invention relates to an anti-IL-6 receptor antibody having excellent antigen-binding activity, neutralizing activity, plasma retention, stability and/or homogeneity, and having a reduced risk of immunogenicity.

The anti-IL-6 receptor antibody is preferably a humanized PM-1 antibody (TOCILIZUMAB). More specifically, the present invention provides humanized PM-1 antibodies with enhanced antigen-binding activity, enhanced neutralization activity, improved plasma retention, reduced immunogenicity risk, improved stability, and improved homogeneity through amino acid substitution.

The humanized PM-1 antibody binds to the human IL-6 receptor and inhibits the binding of human IL-6 to the human IL-6 receptor. In this specification, the amino acid sequence of the humanized PM-1 antibody corresponds to the SEQ ID NO in the sequence listing as follows:

Heavy chain amino acid sequence SEQ ID NO: 15

Light chain amino acid sequence SEQ ID NO: 16

The amino acid sequence of the heavy chain variable region is SEQ ID NO: 17

The amino acid sequence of the light chain variable region is SEQ ID NO: 18

The amino acid sequence of heavy chain CDR1 (HCDR1) is SEQ ID NO: 1

The amino acid sequence of heavy chain CDR2 (HCDR2) is SEQ ID NO: 2

The amino acid sequence of heavy chain CDR3 (HCDR3) is SEQ ID NO: 3

The amino acid sequence of heavy chain FR1 (HFR1) is SEQ ID NO: 7

The amino acid sequence of heavy chain FR2 (HFR2) is SEQ ID NO: 8

The amino acid sequence of heavy chain FR3 (HFR3) is SEQ ID NO: 9

The amino acid sequence of heavy chain FR4 (HFR4) is SEQ ID NO: 10

The amino acid sequence of light chain CDR1 (LCDR1) is SEQ ID NO: 4

The amino acid sequence of light chain CDR2 (LCDR2) is SEQ ID NO: 5

The amino acid sequence of light chain CDR3 (LCDR3) is SEQ ID NO: 6

The amino acid sequence of light chain FR1 (LFR1) is SEQ ID NO: 11

The amino acid sequence of light chain FR2 (LFR2) is SEQ ID NO: 12

The amino acid sequence of light chain FR3 (LFR3) is SEQ ID NO: 13

The amino acid sequence of light chain FR4 (LFR4) is SEQ ID NO: 14

<Antibodies with Enhanced Affinity and Neutralizing Activity>

The present invention provides anti-human IL-6 receptor antibodies having high binding and/or neutralizing activity against the human IL-6 receptor. More specifically, the present invention provides the antibodies described in (a) to (y) below, and methods for producing the antibodies.

(a) An anti-human IL-6 receptor antibody comprising a heavy chain CDR1 in which Ser at position 1 in the amino acid sequence (HCDR1) of SEQ ID NO: 1 is substituted with another amino acid.

The substituted amino acid is not particularly limited, but is preferably substituted with Trp (RD_68), Thr (RD_37), Asp (RD_8), Asn (RD_11), Arg (RD_31), Val (RD_32), Phe (RD_33), Ala (RD_34), Gln (RD_35), Tyr (RD_36), Leu (RD_38), His (RD_42), Glu (RD_45) or Cys (RD_46).

The sequence in which Ser at position 1 in the amino acid sequence of SEQ ID NO: 1 is substituted with Trp is shown in SEQ ID NO: 26.

The sequence in which Ser at position 1 in the amino acid sequence of SEQ ID NO: 1 is substituted with Thr is shown in SEQ ID NO: 27.

The sequence in which Ser at position 1 in the amino acid sequence of SEQ ID NO: 1 is substituted with Asp is shown in SEQ ID NO: 28.

The sequence in which Ser at position 1 in the amino acid sequence of SEQ ID NO: 1 is substituted with Asn is shown in SEQ ID NO: 29.

The sequence in which Ser at position 1 in the amino acid sequence of SEQ ID NO: 1 is substituted with Arg is shown in SEQ ID NO: 30.

The sequence in which Ser at position 1 in the amino acid sequence of SEQ ID NO: 1 is substituted with Val is shown in SEQ ID NO: 31.

The sequence in which Ser at position 1 in the amino acid sequence of SEQ ID NO: 1 is substituted with Phe is shown in SEQ ID NO: 32.

The sequence in which Ser at position 1 in the amino acid sequence of SEQ ID NO: 1 is substituted with Ala is shown in SEQ ID NO: 33.

The sequence in which Ser at position 1 in the amino acid sequence of SEQ ID NO: 1 is substituted with Gln is shown in SEQ ID NO: 34.

The sequence in which Ser at position 1 in the amino acid sequence of SEQ ID NO: 1 is substituted with Tyr is shown in SEQ ID NO: 35.

The sequence in which Ser at position 1 in the amino acid sequence of SEQ ID NO: 1 is substituted with Leu is shown in SEQ ID NO: 36.

The sequence in which Ser at position 1 in the amino acid sequence of SEQ ID NO: 1 is substituted with His is shown in SEQ ID NO: 37.

The sequence in which Ser at position 1 in the amino acid sequence of SEQ ID NO: 1 is substituted with Glu is shown in SEQ ID NO: 38.

The sequence in which Ser at position 1 in the amino acid sequence of SEQ ID NO: 1 is substituted with Cys is shown in SEQ ID NO: 39.

(b) An anti-human IL-6 receptor antibody comprising a heavy chain CDR1 in which Trp at position 5 in the amino acid sequence (HCDR1) of SEQ ID NO: 1 is substituted with another amino acid.

The amino acid after substitution is not particularly limited, but substitution to Ile (RD_9) or Val (RD_30) is preferred.

The sequence in which Trp at position 5 in the amino acid sequence of SEQ ID NO: 1 is substituted with Ile is shown in SEQ ID NO: 40.

The sequence in which Trp at position 5 in the amino acid sequence of SEQ ID NO: 1 is substituted with Val is shown in SEQ ID NO: 41.

(c) An anti-human IL-6 receptor antibody comprising a heavy chain CDR2 in which Tyr at position 1 in the amino acid sequence of SEQ ID NO: 2 (HCDR2) is substituted with another amino acid.

The amino acid after substitution is not particularly limited, but substitution with Phe (RD_82) is preferred.

The sequence in which Tyr at position 1 in the amino acid sequence of SEQ ID NO: 2 is substituted with Phe is shown in SEQ ID NO: 42.

(d) An anti-human IL-6 receptor antibody comprising a heavy chain CDR2 in which Thr at position 8 in the amino acid sequence (HCDR2) of SEQ ID NO: 2 is substituted with another amino acid.

The amino acid after substitution is not particularly limited, but substitution with Arg (RD_79) is preferred.

The sequence in which Thr at position 8 in the amino acid sequence of SEQ ID NO: 2 is substituted with Arg is shown in SEQ ID NO: 43.

(e) An anti-human IL-6 receptor antibody comprising a heavy chain CDR2 in which Thr at position 9 in the amino acid sequence of SEQ ID NO: 2 (HCDR2) is substituted with another amino acid.

The amino acid after substitution is not particularly limited, but substitution to Ser (RD_12) or Asn (RD_61) is preferred.

The sequence in which Thr at position 9 in the amino acid sequence of SEQ ID NO: 2 is substituted with Ser is shown in SEQ ID NO: 44.

The sequence in which Thr at position 9 in the amino acid sequence of SEQ ID NO: 2 is substituted with Asn is shown in SEQ ID NO: 45.

(f) An anti-human IL-6 receptor antibody comprising a heavy chain CDR3 in which Ser at position 1 in the amino acid sequence of SEQ ID NO: 3 (HCDR3) is substituted with another amino acid.

The amino acid after substitution is not particularly limited, but preferably substituted with Ile (RD_2), Val (RD_4), Thr (RD_80), or Leu (RD_5).

The sequence in which Ser at position 1 in the amino acid sequence of SEQ ID NO: 3 is substituted with Ile is shown in SEQ ID NO: 46.

The sequence in which Ser at position 1 in the amino acid sequence of SEQ ID NO: 3 is substituted with Val is shown in SEQ ID NO: 47.

The sequence in which Ser at position 1 in the amino acid sequence of SEQ ID NO: 3 is substituted with Thr is shown in SEQ ID NO: 48.

The sequence in which Ser at position 1 in the amino acid sequence of SEQ ID NO: 3 is substituted with Leu is shown in SEQ ID NO: 49.

(g) An anti-human IL-6 receptor antibody comprising a heavy chain CDR3 in which Leu at position 2 in the amino acid sequence of SEQ ID NO: 3 (HCDR3) is substituted with another amino acid.

The amino acid after substitution is not particularly limited, but preferably substituted with Thr (RD_84).

The sequence in which Leu at position 2 in the amino acid sequence of SEQ ID NO: 3 is substituted with Thr is shown in SEQ ID NO: 50.

(h) An anti-human IL-6 receptor antibody comprising a heavy chain CDR3 in which Thr at position 5 in the amino acid sequence (HCDR3) of SEQ ID NO: 3 is substituted with another amino acid.

The amino acid after substitution is not particularly limited, but preferably it is substituted with Ala (RD_3) or Ile (RD_83). Another preferred substitution is that Thr at position 5 is substituted with Ser (RDC_14H).

The sequence in which Thr at position 5 in the amino acid sequence of SEQ ID NO: 3 is substituted with Ala is shown in SEQ ID NO: 51.

The sequence in which Thr at position 5 in the amino acid sequence of SEQ ID NO: 3 is substituted with Ile is shown in SEQ ID NO: 52.

The sequence in which Thr at position 5 in the amino acid sequence of SEQ ID NO: 3 is substituted with Ser is shown in SEQ ID NO: 53.

(i) An anti-human IL-6 receptor antibody comprising a heavy chain CDR3 in which Ala at position 7 in the amino acid sequence (HCDR3) of SEQ ID NO: 3 is substituted with another amino acid.

The amino acid sequence after substitution is not particularly limited, but substitution to Ser (RD_81) or Val (PF_3H) is preferred.

The sequence in which Ala at position 7 in the amino acid sequence of SEQ ID NO: 3 is substituted with Ser is shown in SEQ ID NO: 54.

The sequence in which Ala at position 7 in the amino acid sequence of SEQ ID NO: 3 is substituted with Val is shown in SEQ ID NO: 55.

(j) An anti-human IL-6 receptor antibody comprising a heavy chain CDR3 in which Met at position 8 in the amino acid sequence (HCDR3) of SEQ ID NO: 3 is substituted with another amino acid.

The amino acid sequence after substitution is not particularly limited, but substitution to Leu (PF_4H) is preferred.

The sequence in which Met at position 8 in the amino acid sequence of SEQ ID NO: 3 is substituted with Leu is shown in SEQ ID NO: 56.

(k) An anti-human IL-6 receptor antibody comprising a heavy chain CDR3 in which Ser at position 1 and Thr at position 5 in the amino acid sequence (HCDR3) of SEQ ID NO: 3 are substituted with other amino acids.

The amino acids after substitution are not particularly limited, but preferably, Ser at position 1 is substituted with Leu, and Thr at position 5 is substituted with Ala (RD_6). Other preferred substitutions include: Ser at position 1 is substituted to Val, and Thr at position 5 is substituted to Ala (RDC_2H); Ser at position 1 is substituted to Ile, and Thr at position 5 is substituted to Ala (RDC_3H); Ser at position 1 is substituted to Thr, and Thr at position 5 is substituted to Ala (RDC_4H); Ser at position 1 is substituted to Val, and Thr at position 5 is substituted to Ile (RDC_5H); Ser at position 1 is substituted to Ile, and Thr at position 5 is substituted to Ile (RDC_6H); Ser at position 1 is substituted to Thr, and Thr at position 5 is substituted to Ile (RDC_7H); and Ser at position 1 is substituted to Leu, and Thr at position 5 is substituted to Ile (RDC_8H).

The sequence in which Ser at position 1 is substituted with Leu and Thr at position 5 is substituted with Ala in the amino acid sequence described in SEQ ID NO: 3 is shown in SEQ ID NO: 57.

The sequence in which Ser at position 1 is substituted with Val and Thr at position 5 is substituted with Ala in the amino acid sequence of SEQ ID NO: 3 is shown in SEQ ID NO: 58.

The sequence in which Ser at position 1 is substituted with Ile and Thr at position 5 is substituted with Ala in the amino acid sequence described in SEQ ID NO: 3 is shown in SEQ ID NO: 59.

The sequence in which Ser at position 1 is substituted with Thr and Thr at position 5 is substituted with Ala in the amino acid sequence described in SEQ ID NO: 3 is shown in SEQ ID NO: 60.

The sequence in which Ser at position 1 is substituted with Val and Thr at position 5 is substituted with Ile in the amino acid sequence described in SEQ ID NO: 3 is shown in SEQ ID NO: 61.

The sequence in which Ser at position 1 and Thr at position 5 of the amino acid sequence described in SEQ ID NO: 3 are substituted with Ile and Ile, is shown in SEQ ID NO: 62.

The sequence in which Ser at position 1 is substituted with Thr and Thr at position 5 is substituted with Ile in the amino acid sequence described in SEQ ID NO: 3 is shown in SEQ ID NO: 63.

The sequence in which Ser at position 1 is substituted with Leu and Thr at position 5 is substituted with Ile in the amino acid sequence described in SEQ ID NO: 3 is shown in SEQ ID NO: 64.

(1) An anti-human IL-6 receptor antibody comprising a heavy chain CDR3 in which Leu at position 2, Ala at position 7, and Met at position 8 in the amino acid sequence (HCDR3) set forth in SEQ ID NO: 3 are substituted with other amino acids.

The amino acids after substitution are not particularly limited, but preferably Leu at position 2 is substituted with Thr, Ala at position 7 is substituted with Val, and Met at position 8 is substituted with Leu (RD_78).

The amino acid sequence of SEQ ID NO: 3 in which Leu at position 2 is substituted with Thr, Ala at position 7 is substituted with Val, and Met at position 8 is substituted with Leu is shown in SEQ ID NO: 65.

(m) An anti-human IL-6 receptor antibody comprising a light chain CDR1 in which Arg at position 1 in the amino acid sequence (LCDR1) of SEQ ID NO: 4 is substituted with another amino acid.

The amino acid sequence after substitution is not particularly limited, but substitution to Phe(RD_18) is preferred.

The sequence in which Arg at position 1 in the amino acid sequence of SEQ ID NO: 4 is substituted with Phe is shown in SEQ ID NO: 66.

(n) An anti-human IL-6 receptor antibody comprising a light chain CDR1 in which Gln at position 4 in the amino acid sequence (LCDR1) of SEQ ID NO: 4 is substituted with another amino acid.

The amino acid sequence after substitution is not particularly limited, but substitution to Arg (RD_26) or Thr (RD_20) is preferred.

The sequence in which Gln at position 4 in the amino acid sequence of SEQ ID NO: 4 is substituted with Arg is shown in SEQ ID NO: 67.

The sequence in which Gln at position 4 in the amino acid sequence of SEQ ID NO: 4 is substituted with Thr is shown in SEQ ID NO: 68.

(o) An anti-human IL-6 receptor antibody comprising a light chain CDR1 in which Tyr at position 9 in the amino acid sequence (LCDR1) of SEQ ID NO: 4 is substituted with another amino acid.

The amino acid after substitution is not particularly limited, but substitution with Phe (RD_73) is preferred.

The sequence in which Tyr at position 9 in the amino acid sequence of SEQ ID NO: 4 is substituted with Phe is shown in SEQ ID NO: 69.

(p) An anti-human IL-6 receptor antibody comprising a light chain CDR1 in which Asn at position 11 in the amino acid sequence (LCDR1) of SEQ ID NO: 4 is substituted with another amino acid.

The amino acid sequence after substitution is not particularly limited, but substitution to Ser (RD_27) is preferred.

The sequence in which Asn at position 11 in the amino acid sequence of SEQ ID NO: 4 is substituted with Ser is shown in SEQ ID NO: 70.

(q) An anti-human IL-6 receptor antibody comprising a light chain CDR2 in which Thr at position 2 in the amino acid sequence (LCDR2) of SEQ ID NO: 5 is substituted with another amino acid.

The amino acid sequence after substitution is not particularly limited, but substitution to Gly is preferred.

The sequence in which Thr at position 2 in the amino acid sequence of SEQ ID NO: 5 is substituted with Gly is shown in SEQ ID NO: 71.

(r) An anti-human IL-6 receptor antibody comprising a light chain CDR3 in which Gln at position 1 in the amino acid sequence (LCDR3) of SEQ ID NO: 6 is substituted with another amino acid.

The amino acid sequence after substitution is not particularly limited, but substitution to Gly (RD_28), Asn (RD_29) or Ser (RDC_15L) is preferred.

The sequence in which Gln at position 1 in the amino acid sequence of SEQ ID NO: 6 is substituted with Gly is shown in SEQ ID NO: 72.

The sequence in which Gln at position 1 in the amino acid sequence of SEQ ID NO: 6 is substituted with Asn is shown in SEQ ID NO: 73.

The sequence in which Gln at position 1 in the amino acid sequence of SEQ ID NO: 6 is substituted with Ser is shown in SEQ ID NO: 74.

(s) An anti-human IL-6 receptor antibody having a light chain CDR3 in which Gly at position 3 in the amino acid sequence of SEQ ID NO: 6 is substituted with another amino acid.

The amino acid sequence after substitution is not particularly limited, but substitution to Ser is preferred.

The sequence in which Gly at position 3 in the amino acid sequence of SEQ ID NO: 6 is substituted with Ser is shown in SEQ ID NO: 75.

(t) An anti-human IL-6 receptor antibody comprising a light chain CDR1 and a light chain CDR3, wherein Tyr at position 9 in the amino acid sequence (LCDR1) of the light chain CDR1 is substituted with another amino acid, and Gly at position 3 in the amino acid sequence (LCDR3) of the light chain CDR3 is substituted with another amino acid.

The substituted amino acid is not particularly limited, but preferably, Tyr at position 9 in the amino acid sequence (LCDR1) described in SEQ ID NO: 4 is substituted with Phe, and preferably, Gly at position 3 in the amino acid sequence (LCDR3) described in SEQ ID NO: 6 is substituted with Ser (RD_72).

(u) An anti-human IL-6 receptor antibody comprising a light chain CDR3 in which Thr at position 5 in the amino acid sequence (LCDR3) of SEQ ID NO: 6 is substituted with another amino acid.

The amino acid after substitution is not particularly limited, but is preferably substituted with Arg (RD_23) or Ser.

The sequence in which Thr at position 5 in the amino acid sequence of SEQ ID NO: 6 is substituted with Arg is shown in SEQ ID NO: 76.

The sequence in which Thr at position 5 in the amino acid sequence of SEQ ID NO: 6 is substituted with Ser is shown in SEQ ID NO: 77.

(v) An anti-IL-6 receptor antibody having a light chain CDR3 in which Gln at position 1 and Thr at position 5 in the amino acid sequence (LCDR3) set forth in SEQ ID NO: 6 are substituted with other amino acids.

The amino acids after substitution are not particularly limited, but preferably Gln at position 1 is substituted with Gly, and Thr at position 5 is substituted with Ser (RD_22). Other preferred substitutions include Gln at position 1 with Gly, and Thr at position 5 with Arg (RDC_11L).

The sequence in which Gln at position 1 is substituted with Gly and Thr at position 5 is substituted with Ser in the amino acid sequence of SEQ ID NO: 6 is shown in SEQ ID NO: 78.

The sequence in which Gln at position 1 is substituted with Gly and Thr at position 5 is substituted with Arg in the amino acid sequence of SEQ ID NO: 6 is shown in SEQ ID NO: 79.

(w) An anti-IL-6 receptor antibody comprising a heavy chain CDR2 and a heavy chain CDR3, wherein Thr at position 9 in the amino acid sequence (HCDR2) set forth in SEQ ID NO: 2 is substituted with another amino acid, and Ser at position 1 and Thr at position 5 in the amino acid sequence (HCDR3) set forth in SEQ ID NO: 3 are substituted with other amino acids.

Preferably, Thr at position 9 in the amino acid sequence (HCDR2) of SEQ ID NO: 2 is substituted with Asn. In the amino acid sequence (HCDR3) of SEQ ID NO: 3, preferred amino acid combinations after substitution of Ser at position 1 and Thr at position 5 are: Leu and Ala (RDC_27H); Val and Ala (RDC_28H); Ile and Ala (RDC_30H); Thr and Ala (RDC_4H); Val and Ile (RDC_29H); Ile and Ile (RDC_32H); Thr and Ile (RDC_7H); and Leu and Ile (RDC_8H).

(x) An antibody comprising two variable regions, wherein one variable region has the heavy chain CDR3 described in (k), and the other variable region has the light chain CDR3 described in (v).

(y) The antibody described in (x), further comprising the heavy chain CDR2 described in (e).

The present invention provides an antibody and a method for producing the antibody, wherein the antibody comprises at least the amino acid substitution described in any one of (a) to (y) above. Therefore, the antibodies of the present invention also include antibodies that, in addition to the amino acid substitution described in any one of (a) to (y) above, further comprise amino acid substitutions other than the amino acid substitutions described in (a) to (y) above. The antibodies of the present invention also include antibodies that combine multiple amino acid substitutions described in any one of (a) to (y) above. As the amino acid substitutions described in (a) to (y) above, there is substitution of the CDR amino acid sequence with other amino acids. As amino acid substitutions other than the amino acid substitutions described in (a) to (y) above, there are, for example, substitutions, deletions, additions and/or insertions of the amino acid sequences of other CDR parts. In addition, there are substitutions, deletions, additions and/or insertions of the amino acid sequences of FRs. There are also substitutions, deletions, additions and/or insertions of the amino acid sequences of constant regions.

The antibodies of the present invention also include antibodies obtained by grafting the high-affinity CDRs discovered in the present invention into any framework other than the humanized PM-1 antibody. The antibodies of the present invention include antibodies obtained by introducing mutations into the framework portion (e.g., see Curr. Opin. Biotechnol. 1994 Aug; 5(4): 428-33)) to obtain antibodies with the original affinity, in which the high-affinity CDRs discovered in the present invention were grafted into any framework other than the humanized PM-1 antibody, resulting in decreased affinity; and antibodies obtained by introducing mutations into the CDR portion (e.g., see US2006/0122377) to obtain antibodies with the original affinity.

In the present invention, it is preferred that the humanized PM-1 antibody undergo any of the amino acid substitutions (a) to (y) above. Humanized PM-1 antibodies that undergo any of the amino acid substitutions (a) to (y) above exhibit high neutralizing activity against the IL-6 receptor. Humanized PM-1 antibodies that undergo any of the amino acid substitutions (a) to (y) above are effective as therapeutic agents for inflammatory diseases such as rheumatoid arthritis that are associated with IL-6.

It should be noted that antibodies comprising any of the amino acid substitutions described in (a) to (y) above can also be described, for example, in the form of (1) or (2) below. Although the description herein is based on the antibody described in (a), the same description can be applied to antibodies described in (b) to (y).

(1) An antibody comprising a heavy chain variable region having a CDR1 having an amino acid sequence in which Ser at position 1 in the amino acid sequence of SEQ ID NO: 1 is substituted with another amino acid.

(2) An antibody comprising an H chain having a CDR1 having an amino acid sequence in which Ser at position 1 in the amino acid sequence of SEQ ID NO: 1 is substituted with another amino acid.

<Antibodies with Enhanced Binding Activity>

The present invention also provides an anti-IL-6 receptor antibody with high IL-6 receptor binding activity. In the present invention, an "anti-IL-6 receptor antibody with high IL-6 receptor binding activity" refers to an antibody having an affinity of 1 nM or less, preferably 0.1 nM or less, and more preferably 0.04 nM or less, as measured under physiological conditions at 37°C. Such anti-IL-6 receptor antibodies with high IL-6 receptor binding activity are believed to have an enhanced ability to neutralize the biological effects of the antigen.

The amino acid substitution introduced into the anti-IL-6 receptor antibody having high IL-6 receptor binding activity of the present invention is not particularly limited, and examples thereof include the amino acid substitutions described above.

The IL-6 receptor is not particularly limited, but is preferably a human IL-6 receptor.

The binding activity can be measured by methods known to those skilled in the art, and for example, it can be measured by Biacore (BIACORE) using SPR.

<Antibodies with Reduced Immunogenicity Risk of CDR Sequences>

The present invention also provides anti-IL-6 receptor antibodies with reduced immunogenicity, particularly humanized PM-1 antibodies. It is believed that the presence of T-cell epitopes that bind to HLA within the antibody sequence increases the immunogenicity of the antibody. Therefore, substitutions within the antibody sequence to remove T-cell epitopes present in the antibody sequence can reduce the risk of immunogenicity.

The present invention provides humanized anti-human IL-6 receptor antibodies, particularly humanized PM-1 light chain variable regions, with reduced immunogenicity, by replacing amino acids in the antibody's amino acid sequence, particularly in its CDR sequence, with other amino acids. The present invention also provides antibodies comprising such light chain variable regions.

More specifically, the present invention provides a light chain CDR2 in which Thr at position 2 in the amino acid sequence (LCDR2) set forth in SEQ ID NO:5 is substituted with another amino acid. The present invention also provides a light chain variable region comprising this light chain CDR2. The present invention also provides an anti-IL-6 receptor antibody comprising this light chain variable region. The amino acid sequence after the substitution is not particularly limited, but the substitution is preferably to Gly. A sequence in which Thr at position 2 in the amino acid sequence set forth in SEQ ID NO:5 is substituted with Gly is shown in SEQ ID NO:71. This amino acid substitution is preferably performed in the light chain variable region of the humanized PM-1 antibody.

<FR and CDR of H53/L28>

The present invention also provides anti-human IL-6 receptor antibodies with improved pharmacokinetics, stability, and/or immunogenicity in plasma. Regarding IgG, it has been found that the half-life in plasma of IgG with the same Fc region is correlated with the pI with a high correlation coefficient. Therefore, when the present inventors attempted to alter the pI of the variable regions of two antibodies against different antigens, they successfully controlled the half-life in plasma without modifying the Fc region, which is unrelated to the antigen type. It is believed that the rate at which antibodies nonspecifically enter endothelial cells depends on the physicochemical Coulomb interaction between the negatively charged cell surface and IgG. By lowering the pI of IgG, the Coulomb interaction is reduced, and the amount of antibody nonspecifically entering endothelial cells is reduced. As a result, by reducing antibody metabolism in endothelial cells, the plasma retention of the antibody can be increased.

Specifically, the present invention provides an anti-human IL-6 receptor antibody having a lowered isoelectric point and increased plasma retention by substituting the amino acid sequence of an anti-IL-6 receptor antibody, particularly a humanized PM-1 antibody. Specifically, according to the Kabat numbering (Kabat EA et al., 1991 Sequences of Proteins of Immunological Interest). NIH), H13 (amino acid at position 13 of SEQ ID NO: 7), H16 (amino acid at position 16 of SEQ ID NO: 7), H43 (amino acid at position 8 of SEQ ID NO: 8), H81 (amino acid at position 16 of SEQ ID NO: 9), H105 (amino acid at position 3 of SEQ ID NO: 10), L18 (amino acid at position 18 of SEQ ID NO: 11), L45 (amino acid at position 11 of SEQ ID NO: 12), L79 (amino acid at position 23 of SEQ ID NO: 13), L107 (amino acid at position 10 of SEQ ID NO: 14), H31 (amino acid at position 1 of SEQ ID NO: 1), L24 (amino acid at position 1 of SEQ ID NO: 4), and/or L53 (amino acid at position 1 of SEQ ID NO: 5) of humanized PM-1. The amino acid at position 4 of SEQ ID NO:5 is substituted with other amino acids that lower the isoelectric point. This allows the isoelectric point to be lowered without affecting the binding activity and stability of the humanized PM-1. Furthermore, in the humanized PM-1 antibody, several amino acid residues are left in the mouse sequence to maintain binding activity when the mouse sequence is humanized. Specifically, in the above-mentioned Kabat numbering, H27 (amino acid at position 27 of SEQ ID NO:7), H28 (amino acid at position 28 of SEQ ID NO:7), H29 (amino acid at position 29 of SEQ ID NO:7), H30 (amino acid at position 30 of SEQ ID NO:7), and H71 in the humanized PM-1 antibody directly utilize the mouse sequence. Regarding HFR1, it is believed that by replacing H13, H16, H23, and H30, HFR1 can be converted to a human sequence, allowing the production of antibodies with a further reduced immunogenicity risk compared to the humanized PM-1 antibody. Furthermore, since the humanized PM-1 antibody is humanized through CDR grafting, it is believed that there is room for improvement in stability. For example, it is believed that by replacing surface-exposed amino acid residues with hydrophilic amino acids in the antibody variable region, the antibody can be stabilized. Furthermore, by converting the CDR sequence to a consensus sequence, the antibody can also be stabilized. Regarding the humanized PM-1 antibody, by replacing Met at H69 (amino acid at position 4 of SEQ ID NO: 9) in the above-mentioned Kabat numbering with Ile (stabilizing the hydrophobic core structure), Leu at H70 (amino acid at position 5 of SEQ ID NO: 9) with Ser (hydrophilizing surface-exposed residues), Thr at H58 (amino acid at position 9 of SEQ ID NO: 2) with Asn (converting the heavy chain CDR2 to a consensus sequence), Ser at H65 (amino acid at position 16 of SEQ ID NO: 2) with Gly (substituting the β-turn portion with Gly and converting the heavy chain CDR2 to a consensus sequence), or Thr at L93 (amino acid at position 5 of SEQ ID NO: 6) with Ser (hydrophilizing surface-exposed residues), the antibody can be stabilized. By substituting Thr at position 2, L51, of LCDR2 (SEQ ID NO: 5), with Gly, the T cell epitope predicted in silico can be removed without affecting binding activity or stability, thereby reducing the risk of immunogenicity. Combining these amino acid substitutions can yield anti-IL-6 receptor antibodies with improved plasma pharmacokinetics, immunogenicity, and stability.

Examples of such antibodies include the antibodies described in any one of the following (1) to (37).

(1) An antibody comprising a heavy chain variable region having FR1, wherein Arg at position 13 in the amino acid sequence of SEQ ID NO: 7 is substituted with another amino acid.

The amino acid sequence after substitution is not particularly limited, but substitution to Lys is preferred.

The sequence in which Arg at position 13 in the amino acid sequence of SEQ ID NO: 7 is substituted with Lys is shown in SEQ ID NO: 80.

(2) An antibody comprising a heavy chain variable region having FR1, wherein Gln at position 16 in the amino acid sequence of SEQ ID NO: 7 is substituted with another amino acid.

The amino acid sequence after substitution is not particularly limited, but substitution to Glu is preferred.

The sequence in which Gln at position 16 in the amino acid sequence of SEQ ID NO: 7 is substituted with Glu is shown in SEQ ID NO: 81.

(3) An antibody comprising a heavy chain variable region having FR1, wherein Thr at position 23 in the amino acid sequence of SEQ ID NO: 7 is substituted with another amino acid.

The amino acid sequence after substitution is not particularly limited, but substitution to Ala is preferred.

The sequence in which Thr at position 23 in the amino acid sequence of SEQ ID NO: 7 is substituted with Ala is shown in SEQ ID NO: 82.

(4) An antibody comprising a heavy chain variable region having FR1, wherein Thr at position 30 in the amino acid sequence of SEQ ID NO: 7 is substituted with another amino acid.

The amino acid sequence after substitution is not particularly limited, but substitution to Ser is preferred.

The sequence in which Thr at position 30 in the amino acid sequence of SEQ ID NO: 7 is substituted with Ser is shown in SEQ ID NO: 83.

(5) An antibody comprising a heavy chain variable region having FR1, wherein Arg at position 13, Gln at position 16, Thr at position 23, and Thr at position 30 in the amino acid sequence of SEQ ID NO: 7 are substituted with other amino acids.

The amino acids after substitution are not particularly limited, but preferably, Arg at position 13 is substituted with Lys, Gln at position 16 is substituted with Glu, Thr at position 23 is substituted with Ala, and Thr at position 30 is substituted with Ser.

The amino acid sequence of SEQ ID NO: 7 in which Arg at position 13 is substituted with Lys, Gln at position 16 is substituted with Glu, Thr at position 23 is substituted with Ala, and Thr at position 30 is substituted with Ser is shown in SEQ ID NO: 84.

(6) An antibody comprising a heavy chain variable region having FR2, wherein Arg at position 8 in the amino acid sequence of SEQ ID NO: 8 is substituted with another amino acid.

The amino acid after substitution is not particularly limited, but substitution with Glu is preferred.

The sequence in which Arg at position 8 in the amino acid sequence of SEQ ID NO: 8 is substituted with Glu is shown in SEQ ID NO: 85.

(7) An antibody comprising a heavy chain variable region having FR3, wherein Met at position 4 in the amino acid sequence of SEQ ID NO: 9 is substituted with another amino acid.

The amino acid after substitution is not particularly limited, but substitution to Ile is preferred.

The sequence in which Met at position 4 in the amino acid sequence of SEQ ID NO: 9 is substituted with Ile is shown in SEQ ID NO: 86.

(8) An antibody comprising a heavy chain variable region having FR3, wherein Leu at position 5 in the amino acid sequence of SEQ ID NO: 9 is substituted with another amino acid.

The amino acid sequence after substitution is not particularly limited, but substitution to Ser is preferred.

The sequence in which Leu at position 5 in the amino acid sequence of SEQ ID NO: 9 is substituted with Ser is shown in SEQ ID NO: 87.

(9) An antibody comprising a heavy chain variable region having FR3, wherein Arg at position 16 in the amino acid sequence of SEQ ID NO: 9 is substituted with another amino acid.

The amino acid after substitution is not particularly limited, but substitution with Lys is preferred.

The sequence in which Arg at position 16 in the amino acid sequence of SEQ ID NO: 9 is substituted with Lys is shown in SEQ ID NO: 88.

(10) An antibody comprising a heavy chain variable region having FR3, wherein Val at position 27 in the amino acid sequence of SEQ ID NO: 9 is substituted with another amino acid.

The amino acid sequence after substitution is not particularly limited, but substitution to Ala is preferred.

The sequence in which Val at position 27 in the amino acid sequence of SEQ ID NO: 9 is substituted with Ala is shown in SEQ ID NO: 89.

(11) An antibody comprising a heavy chain variable region having FR3, wherein Met at position 4, Leu at position 5, Arg at position 16, and Val at position 27 in the amino acid sequence (HFR3) set forth in SEQ ID NO: 9 are substituted with other amino acids.

The amino acids after substitution are not particularly limited, but preferably Met at position 4 is substituted with Ile, Leu at position 5 is substituted with Ser, Arg at position 16 is substituted with Lys, and Val at position 27 is substituted with Ala.

The amino acid sequence of SEQ ID NO: 9 in which Met at position 4 is substituted with Ile, Leu at position 5 is substituted with Ser, Arg at position 16 is substituted with Lys, and Val at position 27 is substituted with Ala is shown in SEQ ID NO: 90.

(12) An antibody comprising a heavy chain variable region having FR4, wherein Gln at position 3 in the amino acid sequence (HFR4) set forth in SEQ ID NO: 10 is substituted with another amino acid.

The amino acid after substitution is not particularly limited, but substitution with Glu is preferred.

The sequence in which Gln at position 3 in the amino acid sequence of SEQ ID NO: 10 is substituted with Glu is shown in SEQ ID NO: 91.

(13) An antibody comprising a light chain variable region having FR1, wherein Arg at position 18 in the amino acid sequence (LFR1) of SEQ ID NO: 11 is substituted with another amino acid.

The amino acid after substitution is not particularly limited, but substitution to Ser is preferred.

The sequence in which Arg at position 18 in the amino acid sequence of SEQ ID NO: 11 is substituted with Ser is shown in SEQ ID NO: 92.

(14) An antibody comprising a light chain variable region having FR2, wherein Lys at position 11 in the amino acid sequence (LFR2) set forth in SEQ ID NO: 12 is substituted with another amino acid.

The amino acid after substitution is not particularly limited, but substitution with Glu is preferred.

The sequence in which Lys at position 11 in the amino acid sequence of SEQ ID NO: 12 is substituted with Glu is shown in SEQ ID NO: 93.

(15) An antibody comprising a light chain variable region having FR3, wherein Gln at position 23 in the amino acid sequence of SEQ ID NO: 13 is substituted with another amino acid.

The amino acid after substitution is not particularly limited, but substitution with Glu is preferred.

The sequence in which Gln at position 23 in the amino acid sequence of SEQ ID NO: 13 is substituted with Glu is shown in SEQ ID NO: 94.

(16) An antibody comprising a light chain variable region having FR3, wherein the Pro at position 24 in the amino acid sequence of SEQ ID NO: 13 is substituted with another amino acid.

The amino acid sequence after substitution is not particularly limited, but substitution to Ala is preferred.

The sequence in which Pro at position 24 in the amino acid sequence of SEQ ID NO: 13 is substituted with Ala is shown in SEQ ID NO: 95.

(17) An antibody comprising a light chain variable region having FR3, wherein Ile at position 27 in the amino acid sequence of SEQ ID NO: 13 is substituted with another amino acid.

The amino acid sequence after substitution is not particularly limited, but substitution to Ala is preferred.

The sequence in which Ile at position 27 in the amino acid sequence of SEQ ID NO: 13 is substituted with Ala is shown in SEQ ID NO: 96.

(18) An antibody comprising a light chain variable region having FR3, wherein Gln at position 23, Pro at position 24, and Ile at position 27 in the amino acid sequence (LFR3) set forth in SEQ ID NO: 13 are substituted with other amino acids.

The amino acids after substitution are not particularly limited, but preferably Gln at position 23 is substituted with Glu, Pro at position 24 is substituted with Ala, and Ile at position 27 is substituted with Ala.

The sequence in which Gln at position 23 is substituted with Glu, Pro at position 24 is substituted with Ala, and Ile at position 27 is substituted with Ala in the amino acid sequence described in SEQ ID NO: 13 is shown in SEQ ID NO: 97.

(19) An antibody comprising a light chain variable region having FR4, wherein Lys at position 10 in the amino acid sequence (LFR4) of SEQ ID NO: 14 is substituted with another amino acid.

The amino acid after substitution is not particularly limited, but substitution with Glu is preferred.

The sequence in which Lys at position 10 in the amino acid sequence of SEQ ID NO: 14 is substituted with Glu is shown in SEQ ID NO: 98.

(20) An antibody comprising a heavy chain variable region having FR4, wherein Ser at position 5 in the amino acid sequence (HFR4) of SEQ ID NO: 10 is substituted with another amino acid.

The amino acid after substitution is not particularly limited, but substitution with Thr is preferred.

The sequence in which Ser at position 5 in the amino acid sequence of SEQ ID NO: 10 is substituted with Thr is shown in SEQ ID NO: 132.

(21) An antibody comprising a heavy chain variable region having FR4, wherein Gln at position 3 and Ser at position 5 in the amino acid sequence (HFR4) described in SEQ ID NO: 10 are substituted with other amino acids.

The amino acids after substitution are not particularly limited, but preferably Gln at position 3 is substituted with Glu, and Ser at position 5 is substituted with Thr.

The sequence in which Gln at position 3 is substituted with Glu and Ser at position 5 is substituted with Thr in the amino acid sequence described in SEQ ID NO: 10 is shown in SEQ ID NO: 133.

(22) An antibody comprising the humanized PM-1 heavy chain variable region with amino acid substitutions as described in (5), (6), (11) and (21).

(23) An antibody comprising a humanized PM-1 light chain variable region with amino acid substitutions as described in (13), (14), (18) and (19).

(24) An antibody comprising the heavy chain variable region described in (22) and the light chain variable region described in (23).

(25) An antibody comprising a heavy chain variable region having a CDR1, wherein Ser at position 1 in the amino acid sequence (HCDR1) of SEQ ID NO: 1 is substituted with another amino acid.

The amino acid after substitution is not particularly limited, but substitution to Asp is preferred.

The sequence in which Ser at position 1 in the amino acid sequence of SEQ ID NO: 1 is substituted with Asp is shown in SEQ ID NO: 28.

(26) An antibody comprising a heavy chain variable region having a CDR2, wherein Ser at position 16 in the amino acid sequence of SEQ ID NO: 2 is substituted with another amino acid.

The amino acid after substitution is not particularly limited, but substitution with Gly is preferred.

The sequence in which Ser at position 16 in the amino acid sequence of SEQ ID NO: 2 is substituted with Gly is shown in SEQ ID NO: 99.

(27) An antibody comprising a heavy chain variable region having a CDR2, wherein Thr at position 9 and Ser at position 16 in the amino acid sequence (HCDR2) of SEQ ID NO: 2 are substituted with other amino acids.

The amino acids after substitution are not particularly limited, but preferably Thr at position 9 is substituted with Asn, and Ser at position 16 is substituted with Gly.

The sequence in which Thr at position 9 is substituted with Asn and Ser at position 16 is substituted with Gly in the amino acid sequence of SEQ ID NO: 2 is shown in SEQ ID NO: 100.

(28) An antibody comprising a light chain variable region having a CDR1, wherein the Arg at position 1 in the amino acid sequence (LCDR1) of SEQ ID NO: 4 is substituted with another amino acid.

The amino acid after substitution is not particularly limited, but substitution to Gln is preferred.

The sequence in which Arg at position 1 in the amino acid sequence of SEQ ID NO: 4 is substituted with Gln is shown in SEQ ID NO: 101.

(29) An antibody comprising a light chain variable region having a CDR2, wherein Arg at position 4 in the amino acid sequence of SEQ ID NO: 5 is substituted with another amino acid.

The amino acid after substitution is not particularly limited, but substitution with Glu is preferred.

The sequence in which Arg at position 4 in the amino acid sequence of SEQ ID NO: 5 is substituted with Glu is shown in SEQ ID NO: 102.

(30) An antibody comprising a light chain variable region having a CDR2, wherein Thr at position 2 and Arg at position 4 in the amino acid sequence (LCDR2) of CDR2 set forth in SEQ ID NO: 5 are substituted with other amino acids.

The amino acids after substitution are not particularly limited, but preferably Thr at position 2 is substituted with Gly, and Arg at position 4 is substituted with Glu.

The sequence in which Thr at position 2 is substituted with Gly and Arg at position 4 is substituted with Glu in the amino acid sequence of SEQ ID NO: 5 is shown in SEQ ID NO: 103.

(31) An antibody comprising a light chain variable region having a CDR3, wherein Thr at position 5 in the amino acid sequence (LCDR3) of SEQ ID NO: 6 is substituted with another amino acid.

The amino acid after substitution is not particularly limited, but substitution to Ser is preferred.

The sequence in which Thr at position 5 in the amino acid sequence of SEQ ID NO: 6 is substituted with Ser is shown in SEQ ID NO: 77.

(32) An antibody comprising a heavy chain variable region having amino acid substitutions as described in (25) and (27).

(33) An antibody comprising a light chain variable region having amino acid substitutions as described in (28), (30) and (31).

(34) An antibody comprising the heavy chain variable region described in (32) and the light chain variable region described in (33).

(35) An antibody comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 104 (VH of H53/L28).

(36) An antibody comprising a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 105 (VL of H53/L28).

(37) An antibody comprising the heavy chain variable region described in (35) and the light chain variable region described in (36).

It is preferred that the humanized PM-1 antibody be subjected to the amino acid substitution described in any one of (1) to (37) above. The present invention provides an antibody comprising at least the amino acid substitution described in any one of (1) to (37) above, and a method for producing the antibody. Therefore, the antibodies of the present invention also include antibodies comprising, in addition to the amino acid substitution described in any one of (1) to (37) above, amino acid substitutions other than the amino acid substitutions described in (1) to (37) above. The antibodies of the present invention also include antibodies comprising a combination of a plurality of amino acid substitutions described in any one of (1) to (37) above. Examples of the amino acid substitutions described in (1) to (37) above include substitutions of the amino acid sequences of the FRs and CDRs described above. Examples of amino acid substitutions other than the amino acid substitutions described in (1) to (37) above include substitutions, deletions, additions, and/or insertions of FR and CDR sequences other than those described above. In addition, there are substitutions, deletions, additions, and/or insertions of the amino acid sequences of the constant regions.

In addition to the above-mentioned amino acid modifications, modifications that lower the isoelectric point without reducing the activity of anti-IL-6 receptor antibodies include, for example, substitution of Lys at position 15 and/or Ser at position 16 with other amino acids in the amino acid sequence of SEQ ID NO: 2. The substituted amino acids are not particularly limited, but preferably Lys at position 15 is substituted with Gln, and Ser at position 16 is substituted with Asp. A sequence in which Lys at position 15 is substituted with Gln, and Ser at position 16 is substituted with Asp in the amino acid sequence of SEQ ID NO: 2 is shown in SEQ ID NO: 121. Furthermore, the above-mentioned amino acid substitutions can be made to the amino acid sequence of SEQ ID NO: 100. A sequence in which Lys at position 15 is substituted with Gln, and Gly at position 16 is substituted with Asp in the amino acid sequence of SEQ ID NO: 100 is shown in SEQ ID NO: 122. Therefore, the present invention provides an antibody comprising a heavy chain variable region having a CDR2 in which Lys at position 15 and/or Ser at position 16 in the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 100 is substituted with other amino acids.

As another modification to lower the isoelectric point, there is a modification in which Gln at position 4 in the amino acid sequence recorded in SEQ ID NO: 4 is replaced by another amino acid. The substituted amino acid is not particularly limited, but is preferably replaced by Glu. The amino acid sequence in which Gln at position 4 in the amino acid sequence recorded in SEQ ID NO: 4 is replaced by Glu is shown in SEQ ID NO: 123. The above-mentioned amino acid substitution can be performed on the amino acid sequence of SEQ ID NO: 101. The amino acid sequence in which Gln at position 4 in the amino acid sequence recorded in SEQ ID NO: 101 is replaced by Glu is shown in SEQ ID NO: 124. Therefore, the present invention provides an antibody comprising a light chain variable region having CDR1, wherein Gln at position 4 in the amino acid sequence recorded in SEQ ID NO: 4 or SEQ ID NO: 101 is replaced by another amino acid.

In addition, as other modifications to lower the isoelectric point, an example is a modification in which His at position 6 in the amino acid sequence recorded in SEQ ID NO: 5 is replaced by another amino acid. The substituted amino acid is not particularly limited, but is preferably replaced by Glu. The amino acid sequence in which His at position 6 in the amino acid sequence recorded in SEQ ID NO: 5 is replaced by Glu is shown in SEQ ID NO: 125. In addition, the above-mentioned amino acid substitution can be performed on the amino acid sequence of SEQ ID NO: 103. The amino acid sequence in which His at position 6 in the amino acid sequence recorded in SEQ ID NO: 103 is replaced by Glu is shown in SEQ ID NO: 126. Therefore, the present invention provides an antibody comprising a light chain variable region having CDR2, wherein His at position 6 in the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 103 is replaced by another amino acid.

Furthermore, in the amino acid sequence of the heavy chain FR3 described in SEQ ID NO:90, an example of a modification that reduces the risk of immunogenicity is a substitution of Ala at position 27 (Kabat numbering H89) with Val. The amino acid sequence in which Ala at position 27 in the amino acid sequence described in SEQ ID NO:90 is substituted with Val is shown in SEQ ID NO:127. Therefore, the present invention provides an antibody comprising a heavy chain variable region having FR3 in which Ala at position 27 in the amino acid sequence of SEQ ID NO:90 is substituted with Val.

The only mouse sequence remaining in the amino acid sequence of the heavy chain FR3 set forth in SEQ ID NO: 9 or SEQ ID NO: 90 is an Arg at position 6 (Kabat numbering H71). It is believed that by using a human sequence from the human VH1 subclass (SEQ ID NO: 128) or human VH3 subclass (SEQ ID NO: 129) retaining the Arg at position H71 as the FR3 sequence, an anti-human IL-6 receptor antibody with a completely human framework can be prepared. Therefore, the present invention provides an antibody comprising a heavy chain variable region having a FR3 set forth in SEQ ID NO: 128 or SEQ ID NO: 129.

Furthermore, in the amino acid sequence of the heavy chain FR4 described in SEQ ID NO: 10, an example of a modification to improve stability is a substitution of Ser at position 5 (Kabat numbering H107) with Ile. The amino acid sequence in which Ser at position 5 is substituted with Ile in the amino acid sequence described in SEQ ID NO: 10 is shown in SEQ ID NO: 130. Alternatively, this amino acid can be introduced into the amino acid sequence described in SEQ ID NO: 91. The amino acid sequence in which Ser at position 5 is substituted with Ile in the amino acid sequence described in SEQ ID NO: 91 is shown in SEQ ID NO: 131. Therefore, the present invention provides an antibody comprising a heavy chain variable region having FR4 in which Ser at position 5 is substituted with Ile in the amino acid sequence described in SEQ ID NO: 10 or SEQ ID NO: 91.

The above amino acid substitutions are preferably made to humanized PM-1 antibody, H53/L28 (an antibody comprising a heavy chain variable region of SEQ ID NO: 104 and a light chain variable region of SEQ ID NO: 105), or PF1 antibody (an antibody comprising a heavy chain variable region of SEQ ID NO: 22 and a light chain variable region of SEQ ID NO: 23). The present invention provides antibodies comprising at least the above amino acid substitutions and methods for producing the antibodies. Therefore, the antibodies of the present invention also include antibodies comprising, in addition to the above amino acid substitutions, the amino acid substitutions described in (1) to (37) above and/or amino acid substitutions other than those described in (1) to (37) above. Examples of amino acid substitutions other than those described in (1) to (37) above include substitutions, deletions, additions, and/or insertions of FR and CDR sequences other than those described above. In addition, substitutions, deletions, additions, and/or insertions of the amino acid sequence of the constant region are also included.

<Low isoelectric point anti-human IL-6 receptor antibody>

The present invention also provides anti-IL-6 receptor antibodies with low isoelectric points. The low isoelectric point antibodies of the present invention include antibodies with low measured isoelectric points of full-length antibodies and antibodies with low theoretical isoelectric points of variable regions (VH/VL).

In the present invention, an anti-IL-6 receptor antibody having a low measured isoelectric point of the full-length antibody generally refers to an antibody having a measured isoelectric point of 7.5 or less, preferably an antibody having a measured isoelectric point of 7.0 or less, and more preferably an antibody having a measured isoelectric point of 6.0 or less. The measured isoelectric point can be determined by methods known to those skilled in the art, for example, by native gel isoelectric electrophoresis or capillary isoelectric electrophoresis.

In the present invention, an anti-IL-6 receptor antibody having a low theoretical isoelectric point in the variable region generally refers to an antibody having a theoretical isoelectric point of 5.5 or less, preferably an antibody having a theoretical isoelectric point of 5.0 or less, and more preferably an antibody having a theoretical isoelectric point of 4.0 or less. The theoretical isoelectric point can be calculated by methods known to those skilled in the art, for example, by using software such as GENETYX (GENETYX CORPORATION).

The amino acid substitutions introduced into the low isoelectric point anti-IL-6 receptor antibodies of the present invention are not particularly limited, and examples thereof include the above-mentioned amino acid substitutions. Such low isoelectric point anti-IL-6 receptor antibodies are believed to have improved plasma retention.

The IL-6 receptor is not particularly limited, but is preferably a human IL-6 receptor.

<Anti-human IL-6 receptor antibody stable at high concentrations>

The present invention also provides an anti-IL-6 receptor antibody that is stable at high concentrations.

In the present invention, "stable at high concentrations" means that, under appropriately selected buffer conditions within the pH range of 6.5 to 7.0 suitable for subcutaneous administration (e.g., 20 mM histidine-HCl, 150 mM NaCl), the increase in the aggregate ratio (aggregate peak area in gel filtration chromatography/total peak area × 100) of a 100 mg/mL high-concentration anti-IL-6 receptor antibody solution at 25°C over one month is 0.3% or less, preferably 0.2% or less, and more preferably 0.1% or less. The concentration of the anti-IL-6 receptor antibody may be 100 mg/mL or higher, and may be, for example, 200 mg/mL or 300 mg/mL.

The anti-IL-6 receptor antibody of the present invention that is stable at high concentrations is not particularly limited, and the antibody can be prepared, for example, by utilizing the above-mentioned amino acid substitutions.

The IL-6 receptor is not particularly limited, but is preferably a human IL-6 receptor.

The present invention also provides an antibody wherein the amino acid substitutions of any one of (1) to (37) above are further substituted with the amino acid of any one of (a) to (y) above to enhance binding activity and/or neutralizing activity. One embodiment of such an antibody includes, but is not limited to, an antibody (PF1) comprising a heavy chain variable region having an amino acid sequence set forth in SEQ ID NO: 22 (PF1_H) and a light chain variable region having an amino acid sequence set forth in SEQ ID NO: 23 (PF1_L).

The present invention also provides an anti-IL-6 receptor antibody according to any one of (A) to (I) below:

(A) a heavy chain variable region comprising a CDR1, CDR2, and CDR3, wherein the CDR1 has the amino acid sequence set forth in SEQ ID NO: 165 (CDR1 of VH5-M83), the CDR2 has the amino acid sequence set forth in SEQ ID NO: 166 (CDR2 of VH5-M83), and the CDR3 has the amino acid sequence set forth in SEQ ID NO: 167 (CDR3 of VH5-M83);

(B) a light chain variable region comprising a CDR1, CDR2, and CDR3, wherein the CDR1 has the amino acid sequence set forth in SEQ ID NO: 101 (CDR1 of VL5), the CDR2 has the amino acid sequence set forth in SEQ ID NO: 168 (CDR2 of VL5), and the CDR3 has the amino acid sequence set forth in SEQ ID NO: 79 (CDR3 of VL5);

(C) an antibody comprising the heavy chain variable region of (A) and the light chain variable region of (B);

(D) a heavy chain variable region comprising a CDR1, CDR2, and CDR3, wherein the CDR1 has the amino acid sequence set forth in SEQ ID NO: 169 (CDR1 of VH3-M73), the CDR2 has the amino acid sequence set forth in SEQ ID NO: 170 (CDR2 of VH3-M73), and the CDR3 has the amino acid sequence set forth in SEQ ID NO: 171 (CDR3 of VH3-M73);

(E) a light chain variable region comprising a CDR1, CDR2, and CDR3, wherein the CDR1 has the amino acid sequence set forth in SEQ ID NO: 172 (CDR1 of VL3), the CDR2 has the amino acid sequence set forth in SEQ ID NO: 173 (CDR2 of VL3), and the CDR3 has the amino acid sequence set forth in SEQ ID NO: 79 (CDR3 of VL3);

(F) an antibody comprising the heavy chain variable region described in (D) and the light chain variable region described in (E);

(G) a heavy chain variable region comprising a CDR1, CDR2, and CDR3, wherein the CDR1 has the amino acid sequence set forth in SEQ ID NO: 169 (CDR1 of VH4-M73), the CDR2 has the amino acid sequence set forth in SEQ ID NO: 174 (CDR2 of VH4-M73), and the CDR3 has the amino acid sequence set forth in SEQ ID NO: 171 (CDR3 of VH4-M73);

(H) a light chain variable region comprising CDR1, CDR2, and CDR3, wherein the CDR1 has the amino acid sequence set forth in SEQ ID NO: 175 (CDR1 of VL1), the CDR2 has the amino acid sequence set forth in SEQ ID NO: 173 (CDR2 of VL1), and the CDR3 has the amino acid sequence set forth in SEQ ID NO: 79 (CDR3 of VL1);

(I) An antibody comprising the heavy chain variable region described in (G) and the light chain variable region described in (H).

The present invention also provides an anti-IL-6 receptor antibody according to any one of the following (a) to (q):

(a) an antibody comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 159 (H96-IgG1 variable region);

(b) an antibody comprising a heavy chain variable region having an amino acid sequence in which at least one of the amino acids at position 35: Trp, position 51: Tyr, position 63: Ser, position 65: Lys, position 66: Gly, position 99: Val, position 103: Ile, position 108: Tyr, position 111: Glu, and position 113: Thr in the amino acid sequence of SEQ ID NO: 159 (H96-IgG1 variable region) is substituted with another amino acid;

(c) an antibody comprising a heavy chain variable region having an amino acid sequence in which Lys at position 65, Gly at position 66, Val at position 99, Ile at position 103, Glu at position 111, and Thr at position 113 in the amino acid sequence of SEQ ID NO: 159 (H96-IgG1 variable region) are substituted with other amino acids;

(d) an antibody comprising a heavy chain variable region having an amino acid sequence in which Trp at position 35, Tyr at position 51, Ser at position 63, Lys at position 65, Gly at position 66, Val at position 99, Ile at position 103, and Tyr at position 108 in the amino acid sequence of SEQ ID NO: 159 (H96-IgG1 variable region) are substituted with other amino acids;

(e) an antibody comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 160 (F2H-IgG1 variable region);

(f) an antibody comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 161 (VH5-M83 variable region);

(g) an antibody having a light chain variable region having the following amino acid sequence: an amino acid sequence in which Gln at position 27 and/or His at position 55 in the amino acid sequence (PF1L) set forth in SEQ ID NO: 23 is substituted with other amino acids;

(h) an antibody comprising a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 162 (L39 variable region);

(i) an antibody comprising a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 163 (VL5-κ variable region);

(j) an antibody comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 176 (VH3-M73 variable region);

(k) an antibody comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 178 (VH4-M73 variable region);

(1) an antibody comprising a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 177 (VL3-κ variable region);

(m) an antibody comprising a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 179 (VL1-κ variable region);

(n) an antibody comprising the heavy chain variable region of (e) and the light chain variable region of (h);

(o) an antibody comprising the heavy chain variable region of (f) and the light chain variable region of (i) (a combination of variable regions of FV5-M83);

(p) an antibody comprising the heavy chain variable region of (j) and the light chain variable region of (l) (a combination of variable regions of FV4-M73);

(q) an antibody comprising the heavy chain variable region of (k) and the light chain variable region of (m) (a combination of variable regions of FV3-M73).

In the amino acid substitutions in the heavy chain variable region described in (a) to (d) above, the amino acids to be substituted are not particularly limited, but preferably Trp at position 35 is substituted with Val, Tyr at position 51 is substituted with Phe, Ser at position 63 is substituted with Thr, Lys at position 65 is substituted with Gln, Gly at position 66 is substituted with Asp, Val at position 99 is substituted with Leu, Ile at position 103 is substituted with Ala, Tyr at position 108 is substituted with Val, Glu at position 111 is substituted with Gln, and Thr at position 113 is substituted with Ile. In the amino acid substitutions in the light chain variable region described in (g) above, the amino acids to be substituted are not particularly limited, but preferably Gln at position 27 is substituted with Glu, and His at position 55 is substituted with Glu. In addition, amino acid substitutions, deletions, insertions, and/or additions other than the above amino acid substitutions may be performed.

The antibody constant regions of the present invention are not particularly limited, and any constant region can be used. For example, constant regions having native sequences such as IgG1, IgG2, and IgG4, or modified constant regions produced by substitution, deletion, addition, and/or insertion of amino acids in constant regions having native sequences can be used. Examples of modified constant regions include the constant regions described below.

Examples of antibodies using the variable regions of the present invention include the following:

(1) an antibody comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO: 134 (H96-IgG1);

(2) an antibody comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO: 135 (F2H-IgG1);

(3) an antibody comprising a heavy chain having the amino acid sequence (VH5-IgG1) set forth in SEQ ID NO: 137;

(4) an antibody comprising a heavy chain having the amino acid sequence (VH5-M83) set forth in SEQ ID NO: 139;

(5) an antibody comprising a light chain having the amino acid sequence (L39) set forth in SEQ ID NO: 136;

(6) an antibody comprising a light chain having the amino acid sequence (VL5-κ) set forth in SEQ ID NO: 138;

(7) an antibody comprising a heavy chain having the amino acid sequence (VH3-M73) set forth in SEQ ID NO: 180;

(8) an antibody comprising a heavy chain having the amino acid sequence (VH4-M73) set forth in SEQ ID NO: 182;

(9) an antibody comprising a light chain having the amino acid sequence (VL3-κ) set forth in SEQ ID NO: 181;

(10) an antibody comprising a light chain having the amino acid sequence (VL1-κ) set forth in SEQ ID NO: 183;

(11) an antibody comprising the heavy chain of (2) and the light chain of (5);

(12) an antibody comprising the heavy chain of (3) and the light chain of (6);

(13) an antibody comprising the heavy chain of (4) and the light chain of (6) (FV5-M83);

(14) an antibody comprising the heavy chain of (7) and the light chain of (9) (FV4-M73);

(15) an antibody comprising the heavy chain of (8) and the light chain of (10) (FV3-M73);

(16) An antibody having an activity equivalent to that of the antibody according to any one of (1) to (15).

Here, "having equivalent activity" means that the binding activity and/or neutralizing activity with the antigen are equivalent. In the present invention, equivalent activity does not necessarily mean the same activity, but means, for example, an activity of 50% or more, preferably 70% or more, and more preferably 90% or more.

The present invention also provides the CDR or FR described in any one of the following (i) to (xxii).

(i) a heavy chain FR1 having the amino acid sequence set forth in SEQ ID NO: 84 (heavy chain FR1 of VH5);

(ii) a heavy chain FR1 having the amino acid sequence set forth in SEQ ID NO: 186 (heavy chain FR1 of VH3 and VH4);

(iii) a heavy chain FR2 having the amino acid sequence set forth in SEQ ID NO: 85 (heavy chain FR2 of VH3, VH4, or VH5);

(iv) a heavy chain FR3 having the amino acid sequence set forth in SEQ ID NO: 184 (heavy chain FR3 of VH3, VH4, or VH5);

(v) a heavy chain FR4 having the amino acid sequence set forth in SEQ ID NO: 133 (heavy chain FR4 of VH3, VH4, or VH5);

(vi) a light chain FR1 having the amino acid sequence set forth in SEQ ID NO: 92 (light chain FR1 of VL1, VL3, and VL5);

(vii) a light chain FR2 having the amino acid sequence set forth in SEQ ID NO: 93 (light chain FR2 of VL1, VL3, and VL5);

(viii) a light chain FR3 having the amino acid sequence set forth in SEQ ID NO: 97 (light chain FR3 of VL1, VL3, and VL5);

(ix) a light chain FR4 having the amino acid sequence set forth in SEQ ID NO: 98 (light chain FR4 of VL1, VL3, or VL5);

(x) a heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 169 (heavy chain CDR1 of VH3 and VH4);

(xi) a heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 165 (heavy chain CDR1 of VH5);

(xii) a heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 170 (heavy chain CDR2 of VH3);

(xiii) a heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 174 (heavy chain CDR2 of VH4);

(xiv) a heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 166 (heavy chain CDR2 of VH5);

(xv) a heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 171 (heavy chain CDR3 of VH3 and VH4);

(xvi) a heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 167 (heavy chain CDR3 of VH5);

(xvii) a light chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 175 (light chain CDR1 of VL1);

(xviii) a light chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 172 (light chain CDR1 of VL3);

(xix) a light chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 101 (light chain CDR1 of VL5);

(xx) light chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 173 (light chain CDR2 of VL1 and VL3);

(xxi) a light chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 168 (light chain CDR2 of VL5);

(xxii) A light chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 79 (light chain CDR3 of VL1, VL3, or VL5).

The antibodies of the present invention also include fragments of antibodies containing any of the above-described amino acid substitutions or modifications thereof. Examples of antibody fragments include Fab, F(ab')2, Fv, or single-chain Fv (scFv) formed by linking H chain and L chain Fv via an appropriate linker, H chain single domains or L chain single domains (e.g., Nat. Biotechnol. 2005 Sep; 23(9): 1126-36), Unibodies (WO2007059782 A1), and SMIPs (WO2007014278 A2). The source of the antibody is not particularly limited and may be human, mouse, rat, or rabbit antibodies. The antibodies of the present invention may also be chimeric, humanized, or fully humanized antibodies.

Specifically, antibodies can be treated with enzymes such as papain and pepsin to generate antibody fragments; alternatively, genes encoding the antibody fragments can be constructed, introduced into expression vectors, and then expressed in appropriate host cells (see, for example, Co, M.S. et al., J. Immunol. (1994) 152, 2968-2976; Better, M. & Horwitz, A.H. Methods in Enzymology (1989) 178, 476-496; Pluckthun, A. & Skerra, A. Methods in Enzymology (1989) 178, 497-515; Lamoyi, E., Methods in Enzymology (1989) 121, 652-663; Rousseaux, J. et al., Methods in Enzymology (1989) 121, 652-663). Enzymology (1989) 121, 663-66; Bird, R.E. et al., TIBTECH (1991) 9, 132-137).

scFv is obtained by linking the H chain V region and the L chain V region of an antibody. In this scFv, the H chain V region and the L chain V region are linked via a linker, preferably a peptide linker (Huston, J.S. et al., Proc. Natl. Acad. Sci. USA (1988) 85, 5879-5883). The H chain V region and the L chain V region in the scFv can be derived from any of the above antibodies. As a peptide linker linking the V regions, for example, any single-chain peptide consisting of 12 to 19 amino acid residues can be used.

<Antibody Constant Region>

The present invention also provides an antibody constant region modified by amino acid substitution as described in any one of the following (i) to (xxi). The constant region refers to an IgG1, IgG2, or IgG4 type constant region. The amino acid sequences of the human IgG1 constant region, the human IgG2 constant region, and the human IgG4 constant region are well known (see SEQ ID NO: 19 for the human IgG1 constant region; SEQ ID NO: 20 for the human IgG2 constant region; and SEQ ID NO: 21 for the human IgG4 constant region). It should be noted that the human IgG4 constant region refers to a sequence into which a modification for improving the stability of the hinge portion (Mol. Immunol. 1993 Jan; 30(1): 105-8) has been introduced. The present invention also provides an antibody comprising an antibody constant region in which the amino acids are substituted. The antibody constant region is preferably a human antibody constant region.

It should be noted that the antibody constant region with amino acid substitutions of the present invention may contain other amino acid substitutions or modifications as long as it contains the amino acid substitutions described in any one of the following (i) to (xxi). Therefore, in the present invention, when the amino acid substitutions of the present invention are made to the IgG2 constant region in which one or more amino acids in the amino acid sequence described in SEQ ID NO: 20 have been substituted and/or modified, or when one or more amino acids are substituted and/or modified after the amino acid substitutions of the present invention have been made, the IgG2 constant region having the amino acid sequence described in SEQ ID NO: 20 corresponds to the IgG2 constant region with amino acid substitutions of the present invention. The same applies to the IgG1 constant region having the amino acid sequence described in SEQ ID NO: 19 and the IgG4 constant region having the amino acid sequence described in SEQ ID NO: 21.

The sugar chain at position 297 in EU numbering (see Sequences of proteins of immunological interest, NIH Publication No. 91-3242) may have any sugar chain structure and may not contain sugar chains (e.g., the constant region produced in host cells such as Escherichia coli without added sugar chains).

(i) Improve the stability of the IgG2 constant region under acidic conditions

One embodiment of the amino acid-substituted IgG2 constant region of the present invention is an IgG2 constant region in which Met at position 276 (EU numbering position 397) in the amino acid sequence of SEQ ID NO: 20 is substituted with another amino acid. The substituted amino acid is not particularly limited, but is preferably substituted with Val. By substituting Met at position 276 (EU numbering position 397) in the amino acid sequence of SEQ ID NO: 20 with another amino acid, the stability of the antibody under acidic conditions can be improved.

(ii) Improving the heterogeneity of the IgG2 constant region

One embodiment of the amino acid substituted IgG2 constant region of the present invention is an IgG2 constant region in which Cys at position 14 (EU numbering position 131), Arg at position 16 (EU numbering position 133), and Cys at position 102 (EU numbering position 219) are substituted with other amino acids in the IgG2 constant region having the amino acid sequence set forth in SEQ ID NO: 20. The substituted amino acids are not particularly limited, but Cys at position 14 (EU numbering position 131) is preferably substituted with Ser, Arg at position 16 (EU numbering position 133) is preferably substituted with Lys, and Cys at position 102 (EU numbering position 219) is preferably substituted with Ser (IgG2-SKSC).

By making these substitutions, heterogeneity in the hinge region from IgG2 can be reduced. The amino acid-substituted IgG2 constant regions of the present invention include those in which at least one of the three amino acid substitutions described above is substituted. However, preferably, Cys at position 14 and Cys at position 102 are substituted with other amino acids, or all three of the above amino acids are substituted.

(iii) Reduce the binding of the IgG2 constant region to FcγR

One embodiment of the amino acid-substituted IgG2 constant region of the present invention provides an IgG2 constant region having an amino acid sequence as set forth in SEQ ID NO: 20, wherein Ala at position 209 (EU330) is substituted with Ser, Pro at position 210 (EU331) is substituted with Ser, and/or Thr at position 218 (EU339) is substituted with Ala. It has been reported that substitution of Ala at position 209 (EU330) or Pro at position 210 (EU331) can reduce the binding of the IgG2 constant region to Fcγ receptors (Eur. J. Immunol. 1999 Aug; 29(8): 2613-24). However, such modifications are not preferred from the perspective of immunogenicity risk because they involve the presence of non-human peptides that can form T cell epitopes. Therefore, by simultaneously substituting Thr at position 218 (EU339) with Ala, the binding of IgG2 to Fcγ receptors can be reduced while using only a human-derived peptide of 9 to 12 amino acids capable of forming a T cell epitope.

The amino acid substituted IgG2 constant region of the present invention only requires that at least one of the three amino acid substitutions described above is substituted, but preferably all three amino acids are substituted. Therefore, a preferred embodiment of the amino acid substituted IgG2 constant region of the present invention is an IgG2 constant region having the amino acid sequence set forth in SEQ ID NO: 20, wherein Ala at position 209 (EU330) is substituted with Ser, Pro at position 210 (EU331) is substituted with Ser, and Thr at position 218 (EU339) is substituted with Ala.

(iv) Improving C-terminal heterogeneity of the IgG2 constant region

The present invention provides an IgG2 constant region in which both the Gly at position 325 (EU numbering position 446) and the Lys at position 326 (EU numbering position 447) are deleted in the IgG2 constant region having the amino acid sequence set forth in SEQ ID NO: 20. By deleting both of these amino acids, heterogeneity originating from the C-terminus of the antibody H chain can be reduced for the first time.

(v) Improving plasma retention by modifying the IgG2 constant region

One embodiment of the amino acid substituted IgG2 constant region of the present invention is: in the IgG2 constant region having the amino acid sequence of SEQ ID NO: 20, His at position 147 (EU numbering position 268), Arg at position 234 (EU numbering position 355), and Gln at position 298 (EU numbering position 419) are substituted with other amino acids in the IgG2 constant region. By these amino acid substitutions, the plasma retention of the antibody can be improved. The substituted amino acid is not particularly limited, but preferably His at position 147 (EU numbering position 268) is substituted with Gln, Arg at position 234 (EU numbering position 355) is substituted with Gln, and Gln at position 298 (EU numbering position 419) is substituted with Glu. In the amino acid substituted IgG2 constant region of the present invention, an IgG2 constant region in which at least one of the above three amino acid substitutions is substituted is included, but preferably all three amino acids are substituted.

(vi) Improve the stability of the IgG4 constant region under acidic conditions

The present invention provides an IgG4 constant region having an amino acid sequence as described in SEQ ID NO: 21 in which Arg at position 289 (EU numbering position 409) is substituted with another amino acid. The substituted amino acid is not particularly limited, but is preferably substituted with Lys. By replacing Arg at position 289 (EU numbering position 409) in the amino acid sequence as described in SEQ ID NO: 21 with another amino acid, the stability of the antibody under acidic conditions can be improved.

(vii) Improving C-terminal heterogeneity of the IgG4 constant region

The present invention provides an IgG4 constant region in which both Gly at position 326 (EU numbering position 446) and Lys at position 327 (EU numbering position 447) are deleted in the IgG4 constant region having the amino acid sequence set forth in SEQ ID NO: 21. By deleting these two amino acids, heterogeneity originating from the C-terminus of the antibody H chain can be reduced for the first time.

(viii) Improvement of C-terminal heterogeneity of IgG1 constant region

The present invention provides an IgG1 constant region in which both the Gly at position 329 (EU numbering position 446) and the Lys at position 330 (EU numbering position 447) are deleted in the IgG1 constant region having the amino acid sequence set forth in SEQ ID NO: 19. By deleting these two amino acids, heterogeneity originating from the C-terminus of the antibody H chain can be reduced for the first time.

(ix)

The present invention provides an IgG1 constant region having the amino acid sequence of SEQ ID NO: 19, wherein Asn at position 317 (EU numbering position 434) is substituted with another amino acid.

The amino acid after substitution is not particularly limited, but substitution to Ala is preferred.

(x)

The present invention provides an IgG2 constant region having the following amino acid sequence: in the amino acid sequence set forth in SEQ ID NO: 20, Ala at position 209 (EU numbering position 330), Pro at position 210 (EU numbering position 331), Thr at position 218 (EU numbering position 339), Met at position 276 (EU numbering position 397), Cys at position 14 (EU numbering position 131), Arg at position 16 (EU numbering position 133), Cys at position 102 (EU numbering position 219), Glu at position 20 (EU numbering position 137), and Ser at position 21 (EU numbering position 138) are substituted with other amino acids.

The amino acids after substitution are not particularly limited, but preferably Ala at position 209 is substituted with Ser, Pro at position 210 is substituted with Ser, Thr at position 218 is substituted with Ala, Met at position 276 is substituted with Val, Cys at position 14 is substituted with Ser, Arg at position 16 is substituted with Lys, Cys at position 102 is substituted with Ser, Glu at position 20 is substituted with Gly, and Ser at position 21 is substituted with Gly.

(xi)

The present invention provides an IgG2 constant region having the following amino acid sequence: in the amino acid sequence set forth in SEQ ID NO: 20, Ala at position 209 (EU numbering position 330), Pro at position 210 (EU numbering position 331), Thr at position 218 (EU numbering position 339), Met at position 276 (EU numbering position 397), Cys at position 14 (EU numbering position 131), Arg at position 16 (EU numbering position 133), Cys at position 102 (EU numbering position 219), Glu at position 20 (EU numbering position 137), and Ser at position 21 (EU numbering position 138) are substituted with other amino acids, and Gly at position 325 (EU numbering position 446) and Lys at position 326 (EU numbering position 447) are both deleted.

The amino acids after substitution are not particularly limited, but preferably Ala at position 209 is substituted with Ser, Pro at position 210 is substituted with Ser, Thr at position 218 is substituted with Ala, Met at position 276 is substituted with Val, Cys at position 14 is substituted with Ser, Arg at position 16 is substituted with Lys, Cys at position 102 is substituted with Ser, Glu at position 20 is substituted with Gly, and Ser at position 21 is substituted with Gly.

(xii)

The present invention provides an IgG2 constant region having the following amino acid sequence: in the amino acid sequence described in SEQ ID NO: 20, Met at position 276 (EU numbering position 397), Cys at position 14 (EU numbering position 131), Arg at position 16 (EU numbering position 133), Cys at position 102 (EU numbering position 219), Glu at position 20 (EU numbering position 137), and Ser at position 21 (EU numbering position 138) are substituted with other amino acids.

The amino acids after substitution are not particularly limited, but preferably Met at position 276 is substituted with Val, Cys at position 14 is substituted with Ser, Arg at position 16 is substituted with Lys, Cys at position 102 is substituted with Ser, Glu at position 20 is substituted with Gly, and Ser at position 21 is substituted with Gly.

(xiii)

The present invention provides an IgG2 constant region having the following amino acid sequence: in the amino acid sequence set forth in SEQ ID NO: 20, Met at position 276 (EU numbering position 397), Cys at position 14 (EU numbering position 131), Arg at position 16 (EU numbering position 133), Cys at position 102 (EU numbering position 219), Glu at position 20 (EU numbering position 137), and Ser at position 21 (EU numbering position 138) are substituted with other amino acids, and both Gly at position 325 (EU numbering position 446) and Lys at position 326 (EU numbering position 447) are deleted.

The amino acids after substitution are not particularly limited, but preferably Met at position 276 is substituted with Val, Cys at position 14 is substituted with Ser, Arg at position 16 is substituted with Lys, Cys at position 102 is substituted with Ser, Glu at position 20 is substituted with Gly, and Ser at position 21 is substituted with Gly.

(xiv)

The present invention provides an IgG2 constant region having the following amino acid sequence: in the amino acid sequence set forth in SEQ ID NO: 20, Cys at position 14 (EU numbering position 131), Arg at position 16 (EU numbering position 133), Cys at position 102 (EU numbering position 219), Glu at position 20 (EU numbering position 137), Ser at position 21 (EU numbering position 138), His at position 147 (EU numbering position 268), Arg at position 234 (EU numbering position 355), and Gln at position 298 (EU numbering position 419) are substituted with other amino acids, and both Gly at position 325 (EU numbering position 446) and Lys at position 326 (EU numbering position 447) are deleted.

The amino acids after substitution are not particularly limited, but preferably, Cys at position 14 is substituted with Ser, Arg at position 16 is substituted with Lys, Cys at position 102 is substituted with Ser, Glu at position 20 is substituted with Gly, Ser at position 21 is substituted with Gly, His at position 147 is substituted with Gln, Arg at position 234 is substituted with Gln, and Gln at position 298 is substituted with Glu.

(xv)

The present invention provides an IgG2 constant region having the following amino acid sequence: in the amino acid sequence set forth in SEQ ID NO: 20, Cys at position 14 (EU numbering position 131), Arg at position 16 (EU numbering position 133), Cys at position 102 (EU numbering position 219), Glu at position 20 (EU numbering position 137), Ser at position 21 (EU numbering position 138), His at position 147 (EU numbering position 268), Arg at position 234 (EU numbering position 355), Gln at position 298 (EU numbering position 419), and Asn at position 313 (EU numbering position 434) are substituted with other amino acids, and both Gly at position 325 (EU numbering position 446) and Lys at position 326 (EU numbering position 447) are deleted.

The amino acids after substitution are not particularly limited, but preferably, Cys at position 14 is substituted with Ser, Arg at position 16 is substituted with Lys, Cys at position 102 is substituted with Ser, Glu at position 20 is substituted with Gly, Ser at position 21 is substituted with Gly, His at position 147 is substituted with Gln, Arg at position 234 is substituted with Gln, Gln at position 298 is substituted with Glu, and Asn at position 313 is substituted with Ala.

(xvi)

The present invention provides an IgG4 constant region having the following amino acid sequence: in the amino acid sequence set forth in SEQ ID NO: 21, Arg at position 289 (EU numbering position 409), Cys at position 14, Arg at position 16, Glu at position 20, Ser at position 21, Arg at position 97, Ser at position 100, Tyr at position 102, Gly at position 103, Pro at position 104, and Pro at position 105 (EU numbering positions 131, 133, 137, 138, 214, 217, 219, 220, 221, and 222, respectively), Glu at position 113, Phe at position 114, and Leu at position 115 (EU numbering positions 233, 234, and 235, respectively) are substituted with other amino acids, and Gly at position 116 (EU numbering position 236) is deleted.

The amino acid after substitution is not particularly limited, but preferably Cys at position 14 (EU numbering position 131) is substituted with Ser, Arg at position 16 (EU numbering position 133) is substituted with Lys, Glu at position 20 (EU numbering position 137) is substituted with Gly, Ser at position 21 (EU numbering position 138) is substituted with Gly, Arg at position 97 (EU numbering position 214) is substituted with Thr, Ser at position 100 (EU numbering position 217) is substituted with Arg, Tyr at position 102 (EU numbering position 219) is substituted with Tyr. The amino acid residues at position 103 (EU numbering position 220) are substituted with Ser, Gly at position 103 (EU numbering position 220) is substituted with Cys, Pro at position 104 (EU numbering position 221) is substituted with Val, Pro at position 105 (EU numbering position 222) is substituted with Glu, Glu at position 113 (EU numbering position 233) is substituted with Pro, Phe at position 114 (EU numbering position 234) is substituted with Val, Leu at position 115 (EU numbering position 235) is substituted with Ala, and Arg at position 289 (EU numbering position 409) is substituted with Lys.

(xvii)

The present invention provides an IgG4 constant region having the following amino acid sequence: Arg at position 289 (EU numbering position 409), Cys at position 14, Arg at position 16, Glu at position 20, Ser at position 21, Arg at position 97, Ser at position 100, Tyr at position 102, Gly at position 103, Pro at position 104, and Pro at position 105 (EU numbering positions 131, 133, 137, 138, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 70, 71, 72, 73, 74, 75 4, 217, 219, 220, 221, 222), Glu at position 113, Phe at position 114, and Leu at position 115 (EU numbering positions 233, 234, and 235, respectively) are substituted with other amino acids, and Gly at position 116 (EU numbering position 236), Gly at position 326 (EU numbering position 446), and Lys at position 327 (EU numbering position 447) are deleted.

The amino acid after substitution is not particularly limited, but preferably Cys at position 14 (EU numbering position 131) is substituted with Ser, Arg at position 16 (EU numbering position 133) is substituted with Lys, Glu at position 20 (EU numbering position 137) is substituted with Gly, Ser at position 21 (EU numbering position 138) is substituted with Gly, Arg at position 97 (EU numbering position 214) is substituted with Thr, Ser at position 100 (EU numbering position 217) is substituted with Arg, Tyr at position 102 (EU numbering position 219) is substituted with Tyr. The amino acid residues at position 103 (EU numbering position 220) are substituted with Ser, Gly at position 103 (EU numbering position 220) is substituted with Cys, Pro at position 104 (EU numbering position 221) is substituted with Val, Pro at position 105 (EU numbering position 222) is substituted with Glu, Glu at position 113 (EU numbering position 233) is substituted with Pro, Phe at position 114 (EU numbering position 234) is substituted with Val, Leu at position 115 (EU numbering position 235) is substituted with Ala, and Arg at position 289 (EU numbering position 409) is substituted with Lys.

(xviii)

The present invention provides an IgG1 constant region having the following amino acid sequence: an IgG1 constant region having the amino acid sequence set forth in SEQ ID NO: 19, in which Asn at position 317 (EU numbering position 434) is substituted with another amino acid, and Gly at position 329 (EU numbering position 446) and Lys at position 330 (EU numbering position 447) are both deleted.

The amino acid after substitution of Asn at position 317 (EU numbering position 434) is not particularly limited, but substitution with Ala is preferred.

(xix)

In the present invention, preferred embodiments of IgG2 having improved hinge region heterogeneity and/or reduced Fcγ receptor binding activity include the following IgG2.

An antibody comprising an IgG2 constant region comprising the amino acid sequence set forth in SEQ ID NO: 20, wherein Ala at position 209, Pro at position 210, Thr at position 218, Cys at position 14, Arg at position 16, Cys at position 102, Glu at position 20, and Ser at position 21 are substituted with other amino acids.

The amino acid after substitution is not particularly limited, but preferably, Ala at position 209 (EU numbering position 330) is substituted with Ser, Pro at position 210 (EU numbering position 331) is substituted with Ser, Thr at position 218 (EU numbering position 339) is substituted with Ala, Cys at position 14 (EU numbering position 131) is substituted with Ser, Arg at position 16 (EU numbering position 133) is substituted with Lys, Cys at position 102 (EU numbering position 219) is substituted with Ser, Glu at position 20 (EU numbering position 137) is substituted with Gly, and Ser at position 21 (EU numbering position 138) is substituted with Gly.

An example of such an IgG2 constant region is an IgG2 constant region having the amino acid sequence of SEQ ID NO: 191 (M86).

Another preferred embodiment of the IgG2 constant region of the present invention is an IgG2 constant region in which Gly at position 325 and Lys at position 326 are further deleted to reduce C-terminal heterogeneity. An example of such an antibody is an IgG2 having a constant region comprising the amino acid sequence of SEQ ID NO: 192 (M86ΔGK).

(xx)

In the present invention, preferred embodiments of the IgG2 constant region with improved heterogeneity in the hinge region include the following IgG2 constant regions.

An IgG2 constant region having the amino acid sequence of SEQ ID NO: 20, wherein Cys at position 14, Arg at position 16, Cys at position 102, Glu at position 20, and Ser at position 21 are substituted with other amino acids.

The amino acids after substitution are not particularly limited, but preferably, Cys at position 14 (EU numbering position 131) is substituted with Ser, Arg at position 16 (EU numbering position 133) is substituted with Lys, Cys at position 102 (EU numbering position 219) is substituted with Ser, Glu at position 20 (EU numbering position 137) is substituted with Gly, and Ser at position 21 (EU numbering position 138) is substituted with Gly.

An example of such an IgG2 constant region is an IgG2 constant region having the amino acid sequence of SEQ ID NO: 193 (M40).

Another preferred embodiment of the IgG2 constant region of the present invention includes an IgG2 constant region in which the above-mentioned IgG2 constant region is further deleted at Gly at position 325 and Lys at position 326. An example of such an antibody is an IgG2 constant region having the amino acid sequence of SEQ ID NO: 194 (M40ΔGK).

(xxi)M14ΔGK, M17ΔGK, M11ΔGK, M31ΔGK, M58, M73, M83, M86ΔGK, M40ΔGK

The present invention also provides an antibody constant region (M14ΔGK) having the amino acid sequence set forth in SEQ ID NO: 24. The present invention also provides an antibody constant region (M17ΔGK) having the amino acid sequence set forth in SEQ ID NO: 116. The present invention also provides an antibody constant region (M11ΔGK) having the amino acid sequence set forth in SEQ ID NO: 25. The present invention also provides an antibody constant region (M31ΔGK) having the amino acid sequence set forth in SEQ ID NO: 118. The present invention also provides an antibody constant region (M58) having the amino acid sequence set forth in SEQ ID NO: 151. The present invention also provides an antibody constant region (M73) having the amino acid sequence set forth in SEQ ID NO: 153. The present invention also provides an antibody constant region (M83) having the amino acid sequence set forth in SEQ ID NO: 164. The present invention also provides an antibody constant region (M86ΔGK) having the amino acid sequence set forth in SEQ ID NO: 192. The present invention also provides an antibody constant region (M40ΔGK) having the amino acid sequence set forth in SEQ ID NO: 194. The above-mentioned antibody constant region has properties such as reduced Fcγ receptor binding activity, reduced immunogenicity risk, improved stability under acidic conditions, reduced heterogeneity, improved plasma retention, and/or higher stability in formulations compared to IgG1 constant regions, and is therefore an optimized antibody constant region.

The present invention provides an antibody comprising the antibody constant region described in any one of (i) to (xxi) above. As long as it has the above-mentioned antibody constant region, there is no limitation on the type of antigen, the source of the antibody, etc., and it can be any antibody. Examples of preferred antibodies include: antibodies that bind to the IL-6 receptor. Other preferred examples of antibodies include: humanized antibodies. Examples of such antibodies include: antibodies having the variable region of the humanized PM-1 antibody. The above-mentioned amino acid substitutions can be made to the variable region of the humanized PM-1 antibody, and other amino acid substitutions, deletions, additions and/or insertions can also be made. Specific examples of substitutions include: modifications to improve the affinity of the above-mentioned (a) to (y), modifications to lower the isoelectric point of (i) to (viii), modifications to improve the stability of the later-mentioned (α) to (ζ), and modifications to reduce immunogenicity, but are not limited to these.

One embodiment of such an antibody is an antibody (PF1) comprising a heavy chain variable region having the amino acid sequence (PF_1+M14ΔGK) recorded in SEQ ID NO: 113 and a light chain variable region having the amino acid sequence (PF1_L) recorded in SEQ ID NO: 23 (the light chain constant region may be κ, λ, or a modified form thereof), but is not limited thereto.

Furthermore, the above-mentioned antibody constant regions and/or antibody molecules comprising the above-mentioned antibody constant regions can also be bound to antibody-like binding molecules (scaffold molecules), physiologically active peptides, binding peptides, etc. in the form of Fc fusion molecules.

In addition to the methods described in the Examples, the antibodies of the present invention can also be obtained as follows. In one embodiment of obtaining the antibodies of the present invention, one or more amino acid residues are first substituted with another desired amino acid in at least one region selected from the CDR region, FR region, and constant region of an anti-IL-6 receptor antibody known to those skilled in the art. The methods for obtaining anti-IL-6 receptor antibodies known to those skilled in the art are not limited. As a method for substituting one or more amino acid residues with other target amino acids in at least one region selected from the group consisting of CDR regions, FR regions, and constant regions, there is, for example, site-specific mutagenesis (Hashimoto-Gotoh, T., Mizuno, T., Ogasahara, Y., and Nakagawa, M. Anoligonucleotide-directed dual amber method for site-directed mutagenesis. Gene (1995) 152, 271-275; Zoller, M.J., and Smith, M. Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13 vectors. Methods Enzymol. (1983) 100, 468-500; Kramer, W., Drutsa, V., Jansen, H.W., Kramer, B., Pflugfelder, M., and Fritz, H.J. (1984). The gapped duplex DNA approach tooligonucleotide-directed mutation construction. Nucleic Acids Res. 12, 9441-9456; Kramer W., and Fritz H.J. (1987). Oligonucleotide-directed construction of mutations via gapped duplex DNA Methods. Enzymol. 154, 350-367; Kunkel, T.A. (1985) Rapid and efficient site-specific mutagenesis without phenotypic Selection (rapid and efficient site-specific mutagenesis without phenotypic screening). Proc. Natl. Acad. Sci. USA 82, 488-492). This method can be used to replace the desired amino acid in the antibody with another target amino acid. As a method for replacing other amino acids, amino acid substitutions can be made to appropriate frameworks and CDRs using library technologies such as framework shuffling (Mol Immunol. 2007 Apr; 44(11): 3049-60) and CDR repair (US2006/0122377).

As another method for obtaining an antibody, first, an antibody that binds to the IL-6 receptor is obtained according to methods known to those skilled in the art. If the obtained antibody is a non-human animal antibody, it can also be humanized. Thereafter, the obtained antibody is determined to have neutralizing activity according to methods known to those skilled in the art. The binding or neutralizing activity of the antibody can be determined according to the methods described in the Examples, but is not limited to these methods. Subsequently, one or more amino acid residues in at least one region selected from the CDR region, FR region, and constant region of the antibody are substituted with another target amino acid.

More specifically, the present invention relates to a method for producing an antibody comprising the following steps (a) and (b), wherein the antibody has enhanced neutralizing activity, enhanced binding activity, improved stability, or reduced immunogenicity.

(a) expressing a DNA encoding an H chain, wherein one or more amino acid residues in at least one region selected from the CDR region, the FR region, and the constant region of the H chain are substituted with another target amino acid, and a DNA encoding an L chain, wherein one or more amino acid residues in at least one region selected from the CDR region and the FR region of the L chain are substituted with another target amino acid.

(b) a step of recovering the expression product of step (a).

In the production method of the present invention, first, DNA encoding the H chain of an anti-IL-6 receptor antibody mutant, i.e., DNA encoding the H chain in which one or more amino acid residues in at least one region selected from the CDR region, the FR region, and the constant region are substituted with other desired amino acids, and DNA encoding the L chain of an anti-IL-6 receptor antibody, i.e., DNA encoding the L chain in which one or more amino acid residues in at least one region selected from the CDR region and the FR region are substituted with other desired amino acids, are expressed. The DNA encoding the H chain in which one or more amino acid residues in at least one region selected from the CDR region, the FR region, and the constant region are substituted with other desired amino acids can be obtained, for example, by obtaining a portion of the CDR region, the FR region, or the constant region of DNA encoding a wild-type H chain and then introducing appropriate substitutions so that the codons encoding specific amino acids in at least one region selected from the CDR region, the FR region, and the constant region encode the other desired amino acids. As for the DNA encoding the L chain in which one or more amino acid residues in at least one region selected from the CDR region and the FR region are substituted with other target amino acids, it can be obtained, for example, by obtaining the CDR region and/or FR region of the DNA encoding the wild-type L chain and then introducing appropriate substitutions so that the codons encoding specific amino acids in the CDR region and FR region encode other target amino acids.

First, a DNA encoding a protein in which one or more amino acid residues in at least one region selected from the CDR region, FR region, and constant region of a wild-type H chain are substituted with other target amino acids is designed and chemically synthesized. This DNA can also be used to obtain a DNA encoding an H chain in which one or more amino acid residues in at least one region selected from the CDR region, FR region, and constant region are substituted with other target amino acids. Regarding the L chain, a DNA encoding a protein in which one or more amino acid residues in the CDR region and/or FR region of a wild-type L chain are substituted with other target amino acids is also designed and chemically synthesized. This DNA can also be used to obtain a DNA encoding an L chain in which one or more amino acid residues in the CDR region and/or FR region are substituted with other target amino acids.

The types of amino acid substitutions are not limited thereto, and include those described in the present specification.

Furthermore, DNA encoding an H chain in which one or more amino acid residues are substituted with other desired amino acids in at least one region selected from the CDR region, FR region, and constant region can be produced by dividing the DNA into partial DNAs. Examples of combinations of partial DNAs include, but are not limited to, DNA encoding a variable region and DNA encoding a constant region, or DNA encoding a Fab region and DNA encoding an Fc region. DNA encoding an L chain can also be produced by dividing the DNA into partial DNAs.

As a method for expressing the above-mentioned DNA, the following methods are exemplified. For example, DNA encoding the H chain variable region and DNA encoding the H chain constant region are simultaneously integrated into an expression vector to construct an H chain expression vector. Similarly, DNA encoding the L chain variable region and DNA encoding the L chain constant region are simultaneously integrated into an expression vector to construct an L chain expression vector. These H chain and L chain genes can also be integrated into the same vector. As expression vectors, for example, SV40 virus-based vectors, Epstein-Barr virus vectors, and BPV (papilloma virus)-based vectors can be used, but are not limited to these.

Host cells can be co-transformed with the antibody expression vector prepared according to the above method. In addition to the above-mentioned cells such as CHO cells (Chinese Hamster Ovary), microorganisms such as Escherichia coli, yeast, and Bacillus subtilis, or plant and animal cells can be used as host cells (Nature Biotechnology 25, 563-565 (2007); Nature Biotechnology 16, 773-777 (1998); Biochemical and Biophysical Research Communications 255, 444-450 (1999); Nature Biotechnology 23, 1159-1169 (2005); Journal of Virology 75, 2803-2809 (2001); Biochemical and Biophysical Research Communications 308, 94-100 (2003)). Transformation is preferably performed by the lipofection method (R.W.Malone et al., Proc.Natl.Acad.Sci.USA 86, 6077 (1989); P.L.Felgner et al., Proc.Natl.Acad.Sci.USA 84, 7413 (1987)), electroporation, the calcium phosphate method (F.L.Graham & A.J.van der Eb, Virology 52, 456-467 (1973)), DEAE-dextran method, or the like.

When producing antibodies, the expression product obtained in step (a) is then recovered. The expression product can be recovered, for example, by isolating the expression product from the cells or culture medium of the transformant after culturing the transformant. Antibodies can be isolated and purified by appropriate combinations of centrifugation, ammonium sulfate fractionation, salting out, ultrafiltration, Iq, FcRn, Protein A, Protein G columns, affinity chromatography, ion exchange chromatography, and gel filtration chromatography.

It should be noted that those skilled in the art can appropriately prepare the constant region of the present invention according to methods for obtaining antibodies.

The present invention also relates to a method for enhancing the binding activity or neutralization activity of an anti-IL-6 receptor antibody to an IL-6 receptor, comprising at least one step selected from the following steps (A) to (X):

(A) a step of substituting Ser at position 1 in the amino acid sequence (HCDR1) of SEQ ID NO: 1 with another amino acid;

(B) a step of substituting Trp at position 5 in the amino acid sequence (HCDR1) of SEQ ID NO: 1 with another amino acid;

(C) a step of substituting Tyr at position 1 in the amino acid sequence (HCDR2) of SEQ ID NO: 2 with another amino acid;

(D) a step of substituting Thr at position 8 in the amino acid sequence (HCDR2) of SEQ ID NO: 2 with another amino acid;

(E) a step of substituting Thr at position 9 in the amino acid sequence (HCDR2) of SEQ ID NO: 2 with another amino acid;

(F) a step of substituting Ser at position 1 in the amino acid sequence (HCDR3) of SEQ ID NO: 3 with another amino acid;

(G) a step of substituting Leu at position 2 in the amino acid sequence (HCDR3) of SEQ ID NO: 3 with another amino acid;

(H) a step of substituting Thr at position 5 in the amino acid sequence (HCDR3) of SEQ ID NO: 3 with another amino acid;

(I) a step of substituting Ala at position 7 in the amino acid sequence (HCDR3) of SEQ ID NO: 3 with another amino acid;

(J) a step of substituting Met at position 8 in the amino acid sequence (HCDR3) of SEQ ID NO: 3 with another amino acid;

(K) a step of substituting Ser at position 1 and Thr at position 5 in the amino acid sequence (HCDR3) of SEQ ID NO: 3 with other amino acids;

(L) a step of substituting Leu at position 2, Ala at position 7, and Met at position 8 in the amino acid sequence (HCDR3) of SEQ ID NO: 3 with other amino acids;

(M) a step of substituting Arg at position 1 in the amino acid sequence (LCDR1) of SEQ ID NO: 4 with other amino acids;

(N) a step of substituting Gln at position 4 in the amino acid sequence (LCDR1) of SEQ ID NO: 4 with another amino acid;

(O) a step of substituting Tyr at position 9 in the amino acid sequence (LCDR1) of SEQ ID NO: 4 with another amino acid;

(P) a step of substituting Asn at position 11 in the amino acid sequence (LCDR1) of SEQ ID NO: 4 with another amino acid;

(Q) a step of substituting Thr at position 2 in the amino acid sequence (LCDR2) of SEQ ID NO: 5 with another amino acid;

(R) a step of substituting Gln at position 1 in the amino acid sequence (LCDR3) of SEQ ID NO: 6 with another amino acid;

(S) a step of substituting Gly at position 3 in the amino acid sequence (LCDR3) of SEQ ID NO: 6 with another amino acid;

(T) a step of substituting Tyr at position 9 in the amino acid sequence (LCDR1) of SEQ ID NO: 4 with another amino acid, and a step of substituting Gly at position 3 in the amino acid sequence (LCDR3) of SEQ ID NO: 6 with another amino acid;

(U) a step of substituting Thr at position 5 in the amino acid sequence (LCDR3) of SEQ ID NO: 6 with another amino acid;

(V) a step of substituting Gln at position 1 and Thr at position 5 in the amino acid sequence (LCDR3) of SEQ ID NO: 6 with other amino acids;

(W) a step of substituting Thr at position 9 in the amino acid sequence (HCDR2) of SEQ ID NO: 2 with another amino acid, and a step of substituting Ser at position 1 and Thr at position 5 in the amino acid sequence (HCDR3) of SEQ ID NO: 3 with other amino acids;

(X) The steps described in (V) and (W).

In (A) above, the substituted amino acid is not particularly limited as long as the affinity is improved, but preferably substituted with Trp, Thr, Asp, Asn, Arg, Val, Phe, Ala, Gln, Tyr, Leu, His, Glu or Cys.

In the above (B), the amino acid after substitution is not particularly limited as long as the affinity is improved, but substitution with Ile or Val is preferred.

In the above (C), the amino acid after substitution is not particularly limited as long as the affinity is improved, but substitution with Phe is preferred.

In the above (D), the amino acid after substitution is not particularly limited as long as the affinity is improved, but substitution with Arg is preferred.

In the above (E), the amino acid after substitution is not particularly limited as long as the affinity is improved, but substitution with Ser or Asn is preferred.

In the above (F), the amino acid after substitution is not particularly limited as long as the affinity is improved, but substitution with Ile, Val, Thr or Leu is preferred.

In the above (G), the amino acid after substitution is not particularly limited as long as the affinity is improved, but substitution with Thr is preferred.

In (H) above, the amino acid to be substituted is not particularly limited as long as the affinity is improved, but substitution with Ala, Ile, or Ser is preferred. Another preferred substitution is substitution of Thr at position 5 with Ser.

In the above (I), the amino acid after substitution is not particularly limited as long as the affinity is improved, but substitution with Ser or Val is preferred.

In the above (J), the amino acid after substitution is not particularly limited as long as the affinity is improved, but substitution with Leu is preferred.

In the above (K), the amino acids after substitution are not particularly limited as long as the affinity is improved, but preferably Ser at position 1 is substituted with Leu and Thr at position 5 is substituted with Ala. Other preferred substitution examples include: Ser at position 1 is substituted with Val and Thr at position 5 is substituted with Ala; Ser at position 1 is substituted with Ile and Thr at position 5 is substituted with Ala; Ser at position 1 is substituted with Thr and Thr at position 5 is substituted with Ala; Ser at position 1 is substituted with Val and Thr at position 5 is substituted with Ile; Ser at position 1 is substituted with Ile and Thr at position 5 is substituted with Ile; Ser at position 1 is substituted with Thr and Thr at position 5 is substituted with Ile; or Ser at position 1 is substituted with Leu and Thr at position 5 is substituted with Ile.

In the above (L), the amino acids after substitution are not particularly limited as long as the affinity is improved, but preferably Leu at position 2 is substituted with Thr, Ala at position 7 is substituted with Val, and Met at position 8 is substituted with Leu.

In the above (M), the amino acid after substitution is not particularly limited as long as the affinity is improved, but substitution with Phe is preferred.

In the above (N), the amino acid after substitution is not particularly limited as long as the affinity is improved, but substitution with Arg or Thr is preferred.

In the above (O), the amino acid after substitution is not particularly limited as long as the affinity is improved, but substitution with Phe is preferred.

In the above (P), the amino acid after substitution is not particularly limited as long as the affinity is improved, but substitution to Ser is preferred.

In the above (Q), the amino acid after substitution is not particularly limited as long as the affinity is improved, but substitution with Gly is preferred.

In the above (R), the amino acid after substitution is not particularly limited as long as the affinity is improved, but substitution with Gly, Asn or Ser is preferred.

In the above (S), the amino acid after substitution is not particularly limited as long as the affinity is improved, but substitution to Ser is preferred.

In the above (T), the amino acid after substitution is not particularly limited as long as the affinity is improved, but preferably Tyr in the amino acid sequence described in SEQ ID NO: 4 (LCDR1) is substituted with Phe, and Gly in the amino acid sequence described in SEQ ID NO: 6 (LCDR3) is substituted with Ser.

In the above (U), the amino acid after substitution is not particularly limited as long as the affinity is improved, but substitution with Arg or Ser is preferred.

In (V) above, the amino acids after substitution are not particularly limited as long as the affinity is improved, but preferably Gln at position 1 is substituted with Gly and Thr at position 5 is substituted with Ser. Other preferred substitutions include Gln at position 1 with Gly and Thr at position 5 with Arg.

In the above (W), preferably, Thr at position 9 of the amino acid sequence (HCDR2) described in SEQ ID NO: 2 is substituted with Asn. Preferred amino acid combinations after substitution of Ser at position 1 and Thr at position 5 of the amino acid sequence (HCDR3) described in SEQ ID NO: 3 include Leu and Ala, Val and Ala, Ile and Ala, Thr and Ala, Val and Ile, Ile and Ile, Thr and Ile, or Leu and Ile.

In steps (A) to (X) above, the method for performing amino acid substitution is not particularly limited and can be performed, for example, according to the site-specific mutagenesis method described above or the methods described in the Examples. When amino acid substitution is performed in the heavy chain variable region, the amino acid sequence of the original heavy chain variable region before substitution is preferably the amino acid sequence of the heavy chain variable region of humanized PM-1. When amino acid substitution is performed in the light chain variable region, the amino acid sequence of the original light chain variable region before substitution is preferably the amino acid sequence of the light chain variable region of humanized PM-1. In addition, it is preferred that the amino acid substitutions described in steps (A) to (X) above be performed on the humanized PM-1 antibody.

The method of enhancing the binding activity or neutralizing activity of an anti-IL-6 receptor antibody of the present invention only needs to include at least the steps described in any one of (A) to (X) above. That is, the method of the present invention can include two or more of the steps described in any one of (A) to (X) above. Furthermore, as long as the steps described in any one of (A) to (X) above are included, other steps (e.g., substitution, deletion, addition, and/or insertion of amino acids other than those described in (A) to (X) above) may also be included. For example, substitution, deletion, addition, and/or insertion of amino acids in FR sequences may be performed, and substitution, deletion, addition, and/or insertion of amino acids in constant regions may also be performed. Preferably, the above-described amino acid substitutions are performed on the humanized PM-1 antibody.

<Methods for Reducing the Risk of Immunogenicity of Anti-IL-6 Receptor Antibodies>

The present invention also relates to a method for reducing the immunogenicity of an anti-IL-6 receptor antibody, comprising substituting Thr at position 2 in the amino acid sequence (LCDR2) set forth in SEQ ID NO: 5 with Gly. The method of reducing the immunogenicity of an anti-IL-6 receptor antibody of the present invention only needs to comprise substituting Thr at position 2 in the amino acid sequence (LCDR2) set forth in SEQ ID NO: 5 with Gly, and may also comprise other amino acid substitutions. The method for amino acid substitution is not particularly limited and can be performed, for example, by the site-specific mutagenesis method described above or the methods described in the Examples.

The above-mentioned amino acid substitutions are preferably made to the humanized PM-1 antibody or its modified (substituted, deleted, inserted) form.

<Method for Lowering the Isoelectric Point of Anti-IL-6 Receptor Antibodies>

The present invention also relates to a method for lowering the isoelectric point of an anti-IL-6 receptor antibody, which comprises at least one step selected from the following (i) to (xv).

(i) a step of substituting Gln at position 16 in the amino acid sequence (HFR1) of SEQ ID NO: 7 with another amino acid;

(ii) a step of substituting Arg at position 8 in the amino acid sequence (HFR2) of SEQ ID NO: 8 with another amino acid;

(iii) a step of substituting Arg at position 16 in the amino acid sequence (HFR3) of SEQ ID NO: 9 with another amino acid;

(iv) substituting Gln at position 3 in the amino acid sequence (HFR4) of SEQ ID NO: 10 with another amino acid;

(v) a step of substituting Arg at position 18 in the amino acid sequence (LFR1) of SEQ ID NO: 11 with another amino acid;

(vi) substituting Lys at position 11 in the amino acid sequence (LFR2) of SEQ ID NO: 12 with another amino acid;

(vii) a step of substituting Gln at position 23 in the amino acid sequence (LFR3) of SEQ ID NO: 13 with another amino acid;

(viii) substituting Lys at position 10 in the amino acid sequence (LFR4) of SEQ ID NO: 14 with another amino acid;

(ix) a step of substituting Ser at position 1 in the amino acid sequence (HCDR1) of SEQ ID NO: 1 with another amino acid;

(x) a step of substituting Arg at position 1 in the amino acid sequence (LCDR1) of SEQ ID NO: 4 with another amino acid;

(xi) a step of substituting Arg at position 4 in the amino acid sequence (LCDR2) of SEQ ID NO: 5 with another amino acid;

(xii) a step of substituting Arg at position 13 in the amino acid sequence (HFR1) of SEQ ID NO: 7 with another amino acid;

(xiii) a step of substituting Lys at position 15 and/or Ser at position 16 in the amino acid sequence (HFR1) described in SEQ ID NO: 2 or the amino acid sequence described in SEQ ID NO: 100 with other amino acids;

(xiv) a step of substituting Gln at position 4 in the amino acid sequence (LCDR1) of SEQ ID NO: 4 or the amino acid sequence of SEQ ID NO: 101 with another amino acid;

(xv) a step of substituting His at position 6 in the amino acid sequence (LCDR2) of SEQ ID NO: 5 or the amino acid sequence of SEQ ID NO: 103 with another amino acid;

In the above (i), the amino acid after substitution is not particularly limited as long as the isoelectric point is lowered, but substitution with Glu is preferred.

In the above (ii), the amino acid after substitution is not particularly limited as long as the isoelectric point is lowered, but substitution with Glu is preferred.

In the above (iii), the amino acid after substitution is not particularly limited as long as the isoelectric point is lowered, but substitution with Lys is preferred.

In the above (iv), the amino acid after substitution is not particularly limited as long as the isoelectric point is lowered, but substitution with Glu is preferred.

In the above (v), the amino acid after substitution is not particularly limited as long as the isoelectric point is lowered, but substitution to Ser is preferred.

In the above (vi), the amino acid after substitution is not particularly limited as long as the isoelectric point is lowered, but substitution with Glu is preferred.

In the above (vii), the amino acid after substitution is not particularly limited as long as the isoelectric point is lowered, but substitution with Glu is preferred.

In the above (viii), the amino acid after substitution is not particularly limited as long as the isoelectric point is lowered, but substitution with Glu is preferred.

In the above (ix), the amino acid after substitution is not particularly limited as long as the isoelectric point is lowered, but substitution to Asp is preferred.

In the above (x), the amino acid after substitution is not particularly limited as long as the isoelectric point is lowered, but substitution to Gln is preferred.

In the above (xi), the amino acid after substitution is not particularly limited as long as the isoelectric point is lowered, but substitution with Glu is preferred.

In the above (xii), the amino acid after substitution is not particularly limited as long as the isoelectric point is lowered, but substitution with Lys is preferred.

In the above (xiii), the amino acid after substitution is not particularly limited as long as the isoelectric point is lowered, but preferably Lys at position 15 is substituted with Gln, and Ser at position 16 is substituted with Asp.

In the above (xiv), the amino acid after substitution is not particularly limited as long as the isoelectric point is lowered, but substitution with Glu is preferred.

In the above (xv), the amino acid after substitution is not particularly limited as long as the isoelectric point is lowered, but substitution with Glu is preferred.

In steps (i) to (xv) above, the method for amino acid substitution is not particularly limited and can be performed, for example, according to the site-specific mutagenesis method described above or the methods described in the Examples. When amino acid substitutions are made to the heavy chain variable region, the amino acid sequence of the original heavy chain variable region before substitution is preferably the amino acid sequence of the heavy chain variable region of humanized PM-1. When amino acid substitutions are made to the light chain variable region, the amino acid sequence of the original light chain variable region before substitution is preferably the amino acid sequence of the light chain variable region of humanized PM-1. In addition, it is preferred that the amino acid substitutions described in steps (i) to (xv) above be made to the humanized PM-1 antibody.

The method for lowering the isoelectric point of an anti-IL-6 receptor antibody of the present invention only needs to include at least the steps described in any one of (i) to (xv) above. That is, the method of the present invention can include two or more of the steps described in (i) to (xv) above. Furthermore, as long as the method includes any one of the steps described in (i) to (xv) above, it may also include other steps (e.g., substitution, deletion, addition, and/or insertion of amino acids other than those described in (i) to (xv) above). For example, substitution, deletion, addition, and/or insertion of amino acid sequences in the constant region may also be performed.

<Method for Improving the Stability of Anti-IL-6 Receptor Antibodies>

The present invention also relates to a method for improving the stability of an anti-IL-6 receptor antibody, which comprises at least one step selected from the following (α) to (ζ).

(α) a step of substituting Met at position 4 in the amino acid sequence (HFR3) of SEQ ID NO: 9 with another amino acid;

(β) a step of substituting Leu at position 5 in the amino acid sequence (HFR3) of SEQ ID NO: 9 with another amino acid;

(γ) a step of substituting Thr at position 9 in the amino acid sequence (HCDR2) of SEQ ID NO: 2 with another amino acid;

(δ) a step of substituting Thr at position 5 in the amino acid sequence (LCDR3) of SEQ ID NO: 6 with another amino acid;

(ε) a step of substituting Ser at position 16 in the amino acid sequence (HCDR2) of SEQ ID NO: 2 with another amino acid;

(ζ) A step of substituting Ser at position 5 in the amino acid sequence (FR4) of SEQ ID NO: 10 with another amino acid.

In the above (α), the amino acid after substitution is not particularly limited as long as the stability is improved, but substitution to Ile is preferred.

In the above (β), the amino acid after substitution is not particularly limited as long as the stability is improved, but substitution with Ser is preferred.

In the above (γ), the amino acid after substitution is not particularly limited as long as the stability is improved, but substitution with Asn is preferred.

In the above (δ), the amino acid after substitution is not particularly limited as long as the stability is improved, but substitution with Ser is preferred.

In the above (ε), the amino acid after substitution is not particularly limited as long as the stability is improved, but substitution with Gly is preferred.

In the above (ζ), the amino acid after substitution is not particularly limited as long as the stability is improved, but substitution to Ile is preferred.

In steps (α) to (ζ) above, the method for amino acid substitution is not particularly limited and can be performed, for example, according to the site-specific mutagenesis method described above or the methods described in the Examples. When amino acid substitutions are made to the heavy chain variable region, the amino acid sequence of the original heavy chain variable region before substitution is preferably the amino acid sequence of the heavy chain variable region of humanized PM-1. When amino acid substitutions are made to the light chain variable region, the amino acid sequence of the original light chain variable region before substitution is preferably the amino acid sequence of the light chain variable region of humanized PM-1. Furthermore, the amino acid substitutions in steps (α) to (ζ) above are preferably performed on the humanized PM-1 antibody.

The method of improving the stability of an anti-IL-6 receptor antibody of the present invention only needs to include at least the steps described in any one of (α) to (ζ) above. That is, the method of the present invention can include two or more of the steps described in (α) to (ζ) above. Furthermore, as long as the method includes any one of the steps described in (α) to (ζ) above, it may also include other steps (e.g., substitution, deletion, addition, and/or insertion of amino acids other than those described in (α) to (ζ) above). For example, amino acid substitution, deletion, addition, and/or insertion of amino acids in the constant region can be performed.

<Method for Reducing Immunogenicity of Anti-IL-6 Receptor Antibodies>

The present invention also relates to a method for reducing the immunogenicity of an anti-IL-6 receptor antibody, particularly a humanized PM-1 antibody, comprising the steps of substituting Arg at position 13 with Lys, Gln at position 16 with Glu, Thr at position 23 with Ala, and/or Thr at position 30 with Ser in the amino acid sequence set forth in SEQ ID NO:7 (HFR1). The method of reducing the immunogenicity of the anti-IL-6 receptor antibody of the present invention only needs to comprise the step of substituting Thr at position 30 with Ser in the amino acid sequence set forth in SEQ ID NO:7 (HFR1) and may further comprise other amino acid substitutions.

The present invention also relates to a method for reducing the immunogenicity of an anti-IL-6 receptor antibody, particularly a humanized PM-1 antibody, comprising substituting Ala at position 27 of the amino acid sequence (HFR3) set forth in SEQ ID NO:90 with Val. The method of reducing the immunogenicity of the anti-IL-6 receptor antibody of the present invention only requires substituting Ala at position 27 of the amino acid sequence (HFR3) set forth in SEQ ID NO:90 with Val, and may further comprise other amino acid substitutions.

The method for amino acid substitution is not particularly limited, and can be performed, for example, by the above-mentioned site-specific mutagenesis method or the method described in the Examples.

The present invention also relates to a method for reducing the immunogenicity of an anti-IL-6 receptor antibody, particularly a humanized PM-1 antibody, H53/L28 or PF1 antibody, by converting FR3 of the antibody into an FR3 having the amino acid sequence of SEQ ID NO: 128 or SEQ ID NO: 129.

<Method for Improving Antibody Stability under Acidic Conditions>

The present invention also relates to a method for improving the stability of an antibody under acidic conditions, the method comprising: replacing Met at position 276 (EU numbering position 397) in the amino acid sequence (IgG2) recorded in SEQ ID NO: 20 with another amino acid. The method of the present invention for improving the stability of an antibody under acidic conditions can be as long as it includes the step of replacing Met at position 276 (EU numbering position 397) in the amino acid sequence (IgG2) recorded in SEQ ID NO: 20 with another amino acid, and can also include other amino acid substitutions. There is no particular limitation on the substituted amino acid, but it is preferably substituted with Val. There is no particular limitation on the method for amino acid substitution, and for example, it can be performed according to the above-mentioned site-specific mutagenesis method or the method described in the examples.

The antibody to be used is not particularly limited, but is preferably an anti-human IL-6 receptor antibody, and more preferably a humanized PM-1 antibody or a modified (substituted, deleted, inserted) form thereof.

<Method for Improving Heterogeneity from the Hinge Region of the IgG2 Constant Region>

The present invention also relates to a method for improving antibody heterogeneity, comprising the steps of replacing Cys at position 14 (EU numbering position 131) in the amino acid sequence described in SEQ ID NO: 20 (IgG2) with another amino acid, replacing Arg at position 16 (EU numbering position 133) with another amino acid, and/or replacing Cys at position 102 (EU numbering position 219) with another amino acid. The substituted amino acids are not particularly limited, but preferably, Cys at position 14 is substituted with Ser, Arg at position 16 is substituted with Lys, and Cys at position 102 is substituted with Ser. The method for improving antibody heterogeneity of the present invention may further include a step of replacing Cys at position 14 (EU numbering position 131) in the amino acid sequence (IgG2) described in SEQ ID NO: 20, a step of replacing Arg at position 16 (EU numbering position 133), and/or a step of replacing Cys at position 102 (EU numbering position 219), and may further include other amino acid substitutions. The method for amino acid substitution is not particularly limited, and for example, it may be performed according to the site-specific mutagenesis method described above or the method described in the examples. The substituted amino acids may be all of the above three amino acids, or one or two (e.g., positions 14 and 102, etc.) amino acids may be substituted.

The antibody to be used is not particularly limited, but is preferably an anti-human IL-6 receptor antibody, and more preferably a humanized PM-1 antibody or a modified (substituted, deleted, inserted) form thereof.

<Method for Reducing Heterogeneity by Deletion of C-Terminal Amino Acids from IgG2 Constant Region>

The present invention also relates to a method for improving antibody heterogeneity, comprising: deleting Gly at position 325 (EU numbering position 446) and Lys at position 326 (EU numbering position 447) in the IgG2 constant region having the amino acid sequence described in SEQ ID NO: 20. The method for improving antibody heterogeneity of the present invention may further comprise deleting Gly at position 325 (EU numbering position 446) and Lys at position 326 (EU numbering position 447) in the IgG2 constant region having the amino acid sequence described in SEQ ID NO: 20, and may further comprise other amino acid substitutions. The method for amino acid substitution is not particularly limited, and for example, it can be performed according to the above-mentioned site-specific mutagenesis method or the method described in the Examples.

The antibody to be used is not particularly limited, but is preferably an anti-human IL-6 receptor antibody, and more preferably a humanized PM-1 antibody or a modified (substituted, deleted, inserted) form thereof.

<Method for reducing FcγR binding while maintaining the human sequence of the IgG2 constant region>

The present invention also relates to a method for reducing antibody binding to FcγR, comprising: a step of substituting Ala at position 209 (EU330) in an IgG2 constant region having an amino acid sequence as set forth in SEQ ID NO: 20 with Ser, a step of substituting Pro at position 210 (EU331) with Ser, and a step of substituting Thr at position 218 (EU339) with Ala. The method of reducing antibody binding to FcγR of the present invention may comprise the step of substituting Ala at position 209 (EU330) in an IgG2 constant region having an amino acid sequence as set forth in SEQ ID NO: 20 with Ser, a step of substituting Pro at position 210 (EU331) with Ser, and a step of substituting Thr at position 218 (EU339) with Ala, and may further comprise other amino acid substitutions. The method for amino acid substitution is not particularly limited, and for example, it may be performed according to the above-mentioned site-specific mutagenesis method or the method described in the Examples.

<Method for Improving Plasma Retention by Substituting Amino Acids in the IgG2 Constant Region>

The present invention also relates to a method for improving the plasma retention of an antibody, comprising: replacing His at position 147 (EU268), Arg at position 234 (EU355), and/or Gln at position 298 (EU419) in an IgG2 constant region having an amino acid sequence as set forth in SEQ ID NO: 20 with other amino acids. The method of improving the plasma retention of an antibody of the present invention may further comprise other amino acid substitutions as long as it comprises the above steps. The substituted amino acids are not particularly limited, but preferably His at position 147 (EU268) is substituted with Gln, Arg at position 234 (EU355) is substituted with Gln, and Gln at position 298 (EU419) is substituted with Glu.

The present invention also relates to a method for improving the plasma retention of an antibody, comprising: substituting Asn at position 313 (EU434) in an IgG2 constant region having the amino acid sequence of SEQ ID NO: 20 or SEQ ID NO: 151 (M58) with another amino acid. The substituted amino acid is not particularly limited, but is preferably substituted with Ala. The method of improving the plasma retention of an antibody of the present invention only needs to include the above steps and may further include other amino acid substitutions.

The antibody to be used is not particularly limited, but is preferably an anti-human IL-6 receptor antibody, and more preferably a humanized PM-1 antibody or a modified (substituted, deleted, inserted) form thereof.

The present invention also relates to methods for reducing antibody heterogeneity originating from the IgG2 hinge portion, methods for improving antibody stability under acidic conditions, methods for reducing antibody heterogeneity originating from the C-terminus, and/or methods for reducing antibody binding to FcγR (M14ΔGK), each of which comprises performing the following steps (a) to (j) on an IgG2 constant region having the amino acid sequence set forth in SEQ ID NO: 20:

(a) replacing Ala at position 209 (EU numbering position 330) of SEQ ID NO: 20 with Ser;

(b) replacing Pro at position 210 (EU numbering position 331) of SEQ ID NO: 20 with Ser;

(c) a step of substituting Thr at position 218 (EU numbering position 339) of SEQ ID NO: 20 with Ala;

(d) a step of substituting Met at position 276 (EU numbering position 397) of SEQ ID NO: 20 with Val;

(e) replacing Cys at position 14 (EU numbering position 131) of SEQ ID NO: 20 with Ser;

(f) a step of substituting Arg at position 16 (EU numbering position 133) of SEQ ID NO: 20 with Lys;

(g) a step of replacing Cys at position 102 (EU numbering position 219) of SEQ ID NO: 20 with Ser;

(h) a step of substituting Glu at position 20 (EU numbering position 137) of SEQ ID NO: 20 with Gly;

(i) a step of substituting Ser at position 21 (EU numbering position 138) of SEQ ID NO: 20 with Gly; and

(j) A step of deleting Gly at position 325 and Lys at position 326 (positions 446 and 447 according to EU numbering, respectively) of SEQ ID NO: 20.

The method of the present invention may further comprise other steps of amino acid substitution and deletion as long as it comprises the above steps. The method of amino acid substitution and deletion is not particularly limited and may be performed, for example, according to the above-mentioned site-specific mutagenesis method or the method described in the Examples.

The antibody to be used is not particularly limited, but is preferably an anti-human IL-6 receptor antibody, and more preferably a humanized PM-1 antibody or a modified (substituted, deleted, inserted) form thereof.

The present invention also relates to methods for reducing antibody heterogeneity originating from the IgG2 hinge portion, methods for improving antibody stability under acidic conditions, and/or methods for reducing antibody heterogeneity originating from the C-terminus (M31ΔGK), each of which comprises performing the following steps (a) to (g) on an IgG2 constant region having the amino acid sequence set forth in SEQ ID NO: 20:

(a) replacing Met at position 276 (EU numbering position 397) of SEQ ID NO: 20 with Val;

(b) replacing Cys at position 14 (EU numbering position 131) of SEQ ID NO: 20 with Ser;

(c) replacing Arg at position 16 (EU numbering position 133) of SEQ ID NO: 20 with Lys;

(d) replacing Cys at position 102 (EU numbering position 219) of SEQ ID NO: 20 with Ser;

(e) a step of substituting Glu at position 20 (EU numbering position 137) of SEQ ID NO: 20 with Gly;

(f) replacing Ser at position 21 (EU numbering position 138) of SEQ ID NO: 20 with Gly; and

(g) A step of deleting Gly at position 325 and Lys at position 326 (positions 446 and 447 according to EU numbering, respectively) of SEQ ID NO: 20.

The present invention also relates to a method for reducing antibody heterogeneity originating from the IgG2 hinge portion, a method for improving antibody plasma retention, and/or a method for reducing antibody heterogeneity originating from the C-terminus (M58), each of which comprises performing the following steps (a) to (i) on an IgG2 constant region having the amino acid sequence set forth in SEQ ID NO: 20:

(a) replacing Cys at position 14 (EU numbering position 131) of SEQ ID NO: 20 with Ser;

(b) replacing Arg at position 16 (EU numbering position 133) of SEQ ID NO: 20 with Lys;

(c) replacing Cys at position 102 (EU numbering position 219) of SEQ ID NO: 20 with Ser;

(d) replacing Glu at position 20 (EU numbering position 137) of SEQ ID NO: 20 with Gly;

(e) replacing Ser at position 21 (EU numbering position 138) of SEQ ID NO: 20 with Gly;

(f) a step of replacing His at position 147 (EU numbering position 268) of SEQ ID NO: 20 with Gln;

(g) a step of substituting Arg at position 234 (EU numbering position 355) of SEQ ID NO: 20 with Gln;

(h) a step of substituting Gln at position 298 (EU numbering position 419) of SEQ ID NO: 20 into Glu; and

(i) A step of deleting Gly at position 325 and Lys at position 326 (positions 446 and 447 according to EU numbering, respectively) of SEQ ID NO: 20.

The present invention also relates to a method for reducing antibody heterogeneity originating from the IgG2 hinge portion, a method for improving antibody plasma retention, and/or a method for reducing antibody heterogeneity originating from the C-terminus (M73), each of which comprises performing the following steps (a) to (j) on an IgG2 constant region having the amino acid sequence set forth in SEQ ID NO: 20:

(a) replacing Cys at position 14 (EU numbering position 131) of SEQ ID NO: 20 with Ser;

(b) replacing Arg at position 16 (EU numbering position 133) of SEQ ID NO: 20 with Lys;

(c) replacing Cys at position 102 (EU numbering position 219) of SEQ ID NO: 20 with Ser;

(d) a step of substituting Glu at position 20 (EU numbering position 137) of SEQ ID NO: 20 with Gly;

(e) replacing Ser at position 21 (EU numbering position 138) of SEQ ID NO: 20 with Gly;

(f) a step of replacing His at position 147 (EU numbering position 268) of SEQ ID NO: 20 with Gln;

(g) a step of replacing Arg at position 234 (EU numbering position 355) of SEQ ID NO: 20 with Gln;

(h) a step of substituting Gln at position 298 (EU numbering position 419) of SEQ ID NO: 20 with Glu;

(i) a step of substituting Asn at position 313 (EU numbering position 434) of SEQ ID NO: 20 into Ala; and

(j) A step of deleting Gly at position 325 and Lys at position 326 (positions 446 and 447 according to EU numbering, respectively) of SEQ ID NO: 20.

The present invention also relates to methods for reducing antibody heterogeneity originating from the IgG2 hinge portion, methods for reducing antibody heterogeneity originating from the C-terminus, and/or methods for reducing antibody binding to FcγR (M86ΔGK), each of which comprises performing the following steps (a) to (i) on an IgG2 constant region having the amino acid sequence set forth in SEQ ID NO: 20:

(a) a step of substituting Ala at position 209 (EU numbering position 330) of SEQ ID NO: 20 with other amino acids;

(b) a step of substituting Pro at position 210 (EU numbering position 331) of SEQ ID NO: 20 with other amino acids;

(c) a step of substituting Thr at position 218 (EU numbering position 339) of SEQ ID NO: 20 with other amino acids;

(d) a step of substituting Cys at position 14 (EU numbering position 131) of SEQ ID NO: 20 with other amino acids;

(e) a step of substituting Arg at position 16 (EU numbering position 133) of SEQ ID NO: 20 with other amino acids;

(f) a step of substituting Cys at position 102 (EU numbering position 219) of SEQ ID NO: 20 with other amino acids;

(g) a step of substituting Glu at position 20 (EU numbering position 137) of SEQ ID NO: 20 with another amino acid;

(h) a step of substituting Ser at position 21 (EU numbering position 138) of SEQ ID NO: 20 with another amino acid; and

(i) A step of deleting Gly at position 325 and Lys at position 326 (positions 446 and 447 according to EU numbering, respectively) of SEQ ID NO: 20.

The amino acid after substitution is not particularly limited, but preferably, Ala at position 209 (EU numbering position 330) is substituted with Ser, Pro at position 210 (EU numbering position 331) is substituted with Ser, Thr at position 218 (EU numbering position 339) is substituted with Ala, Cys at position 14 (EU numbering position 131) is substituted with Ser, Arg at position 16 (EU numbering position 133) is substituted with Lys, Cys at position 102 (EU numbering position 219) is substituted with Ser, Glu at position 20 (EU numbering position 137) is substituted with Gly, and Ser at position 21 (EU numbering position 138) is substituted with Gly.

The present invention also relates to methods for reducing antibody heterogeneity originating from the IgG2 hinge portion and/or methods for reducing antibody heterogeneity originating from the C-terminus (M40ΔGK), each of which comprises performing the following steps (a) to (f) on an IgG2 constant region having the amino acid sequence set forth in SEQ ID NO: 20:

(a) replacing the Cys at position 14 (EU numbering position 131) of SEQ ID NO: 20 with another amino acid;

(b) a step of substituting Arg at position 16 (EU numbering position 133) of SEQ ID NO: 20 with other amino acids;

(c) a step of substituting Cys at position 102 (EU numbering position 219) of SEQ ID NO: 20 with another amino acid;

(d) substituting Glu at position 20 (EU numbering position 137) of SEQ ID NO: 20 with another amino acid;

(e) a step of substituting Ser at position 21 (EU numbering position 138) of SEQ ID NO: 20 with other amino acids; and

(f) A step of deleting Gly at position 325 and Lys at position 326 (positions 446 and 447 according to EU numbering, respectively) of SEQ ID NO: 20.

The amino acids after substitution are not particularly limited, but preferably, Cys at position 14 (EU numbering position 131) is substituted with Ser, Arg at position 16 (EU numbering position 133) is substituted with Lys, Cys at position 102 (EU numbering position 219) is substituted with Ser, Glu at position 20 (EU numbering position 137) is substituted with Gly, and Ser at position 21 (EU numbering position 138) is substituted with Gly.

The method of the present invention may further comprise other steps of amino acid substitution and deletion as long as it comprises the above steps. The method of amino acid substitution and deletion is not particularly limited and may be performed, for example, according to the above-mentioned site-specific mutagenesis method or the method described in the Examples.

The antibody to be used is not particularly limited, but is preferably an anti-human IL-6 receptor antibody, and more preferably a humanized PM-1 antibody or a modified (substituted, deleted, inserted) form thereof.

<Method for Improving the Stability of IgG4 Constant Region under Acidic Conditions>

The present invention also relates to a method for improving the stability of an antibody under acidic conditions, the method comprising the step of replacing Arg at position 289 (EU numbering position 409) in the IgG4 constant region (Mol. Immunol. 1993 Jan; 30(1): 105-8) having the amino acid sequence recorded in SEQ ID NO: 21 with another amino acid. The method of improving the stability of an antibody under acidic conditions of the present invention only needs to include the step of replacing Arg at position 289 (EU numbering position 409) in the amino acid sequence recorded in SEQ ID NO: 21 (human IgG4 constant region) with another amino acid, and may further include other amino acid substitutions. There is no particular limitation on the substituted amino acid, but it is preferably substituted with Lys. There is no particular limitation on the method for amino acid substitution, and for example, it can be performed according to the above-mentioned site-specific mutagenesis method or the method described in the Examples.

The antibody to be used is not particularly limited, but is preferably an anti-human IL-6 receptor antibody, and more preferably a humanized PM-1 antibody or a modified (substituted, deleted, inserted) form thereof.

<Method for Reducing Heterogeneity by Deletion of C-Terminal Amino Acids from IgG4 Constant Region>

The present invention also relates to a method for improving antibody heterogeneity, comprising deleting Gly at position 326 (EU numbering position 446) and Lys at position 327 (EU numbering position 447) in an IgG4 constant region (Mol. Immunol. 1993 Jan; 30(1): 105-8) having the amino acid sequence set forth in SEQ ID NO: 21. The method for improving antibody heterogeneity of the present invention may comprise deleting Lys at position 327 (EU numbering position 447) and/or Gly at position 326 (EU numbering position 446) in an IgG4 constant region (Mol. Immunol. 1993 Jan; 30(1): 105-8) having the amino acid sequence set forth in SEQ ID NO: 21, and may further comprise other amino acid substitutions. The method for amino acid substitution is not particularly limited, and for example, the method may be performed according to the above-mentioned site-specific mutagenesis method or the method described in the Examples.

The antibody to be used is not particularly limited, but is preferably an anti-human IL-6 receptor antibody, and more preferably a humanized PM-1 antibody or a modified (substituted, deleted, inserted) form thereof.

The present invention also relates to methods for improving the stability of IgG4 under acidic conditions, methods for reducing antibody heterogeneity from the C-terminus, and methods for reducing antibody binding to FcγR (M11ΔGK), each of which comprises subjecting an IgG4 constant region having the amino acid sequence set forth in SEQ ID NO: 21 (Mol. Immunol. 1993 Jan; 30(1): 105-8) to the following steps (a) to (p):

(a) replacing Cys at position 14 (EU numbering position 131) of SEQ ID NO: 21 with Ser;

(b) replacing Arg at position 16 (EU numbering position 133) of SEQ ID NO: 21 with Lys;

(c) a step of substituting Glu at position 20 (EU numbering position 137) of SEQ ID NO: 21 with Gly;

(d) replacing Ser at position 21 (EU numbering position 138) of SEQ ID NO: 21 with Gly;

(e) a step of replacing Arg at position 97 (EU numbering position 214) of SEQ ID NO: 21 with Thr;

(f) replacing Ser at position 100 (EU numbering position 217) of SEQ ID NO: 21 with Arg;

(g) replacing Tyr at position 102 (EU numbering position 219) of SEQ ID NO: 21 with Ser;

(h) a step of substituting Gly at position 103 (EU numbering position 220) of SEQ ID NO: 21 with Cys;

(i) replacing Pro at position 104 (EU numbering position 221) of SEQ ID NO: 21 with Val;

(j) a step of replacing Pro at position 105 (EU numbering position 222) of SEQ ID NO: 21 with Glu;

(k) a step of substituting Glu at position 113 (EU numbering position 233) of SEQ ID NO: 21 with Pro;

(1) a step of replacing Phe at position 114 (EU numbering position 234) of SEQ ID NO: 21 with Val;

(m) a step of substituting Leu at position 115 (EU numbering position 235) of SEQ ID NO: 21 into Ala;

(n) deleting the Gly at position 116 (EU numbering position 236) of SEQ ID NO: 21;

(o) a step of substituting Arg at position 289 (EU numbering position 409) of SEQ ID NO: 21 with Lys; and

(p) A step of deleting Gly at position 236 (EU numbering position 446) and Lys at position 237 (EU numbering position 447) of SEQ ID NO: 21.

The method of the present invention may further comprise other steps of amino acid substitution and deletion as long as it comprises the above steps. The method of amino acid substitution and deletion is not particularly limited and may be performed, for example, according to the above-mentioned site-specific mutagenesis method or the method described in the Examples.

The antibody to be used is not particularly limited, but is preferably an anti-human IL-6 receptor antibody, and more preferably a humanized PM-1 antibody or a modified (substituted, deleted, inserted) form thereof.

<Method for Reducing Heterogeneity by Deletion of C-Terminal Amino Acids from IgG1 Constant Region>

The present invention also relates to a method for improving antibody heterogeneity, comprising deleting Gly at position 329 (EU numbering position 446) and Lys at position 330 (EU numbering position 447) in the IgG1 constant region having the amino acid sequence set forth in SEQ ID NO: 19. The method for improving antibody heterogeneity of the present invention may further comprise deleting Lys at position 330 (EU numbering position 447) and Gly at position 329 (EU numbering position 446) in the IgG1 constant region having the amino acid sequence set forth in SEQ ID NO: 19, and may further comprise other amino acid substitutions. The method for amino acid substitution is not particularly limited, and for example, may be performed according to the site-specific mutagenesis method described above or the method described in the Examples.

<Method for Improving Plasma Retention by Substituting Amino Acids in the IgG1 Constant Region>

The present invention also relates to a method for improving the plasma retention of an antibody, comprising substituting Asn at position 317 (EU434) in the IgG1 constant region having the amino acid sequence set forth in SEQ ID NO: 19 with another amino acid. The substituted amino acid is not particularly limited, but is preferably substituted with Ala. The method of improving the plasma retention of an antibody of the present invention may comprise the aforementioned steps and may further include other amino acid substitutions.

The present invention also relates to a method for improving antibody plasma retention and/or reducing antibody heterogeneity from the C-terminus (M83), comprising performing the following steps (a) to (b) on an IgG1 constant region having the amino acid sequence set forth in SEQ ID NO: 19:

(a) substituting Asn at position 317 (EU 434) of SEQ ID NO: 19 into Ala; and

(b) A step of deleting Lys at position 330 (447th in EU numbering) and Gly at position 329 (446th in EU numbering) of SEQ ID NO: 19.

The antibody to be used is not particularly limited, but is preferably an anti-human IL-6 receptor antibody, and more preferably a humanized PM-1 antibody or a modified (substituted, deleted, inserted) form thereof.

The constant regions of the present invention can be combined with variable regions from any antibody, but are preferably combined with variable regions from anti-human IL-6 receptor antibodies. Examples of anti-human IL-6 receptor antibody variable regions include the variable regions of humanized PM-1 antibodies. The variable regions of humanized PM-1 antibodies may be those without or with the aforementioned amino acid substitutions.

The present invention provides a pharmaceutical composition comprising the antibody of the present invention. The pharmaceutical composition of the present invention is useful for treating diseases associated with IL-6, such as rheumatoid arthritis.

In addition to comprising an antibody, the pharmaceutical composition of the present invention may also be introduced into a pharmaceutically acceptable carrier and prepared according to a known method. For example, it can be prepared in the form of an injection of a sterile solution or suspension agent with water or a pharmaceutically acceptable liquid other than water for parenteral administration. For example, it is considered to be appropriately combined with a pharmacologically acceptable carrier or medium, specifically sterile water or physiological saline, vegetable oil, emulsifier, suspending agent, surfactant, stabilizer, flavoring agent, excipient, vehicle, preservative, adhesive, etc., and mixed in the unit dosage form required by the generally recognized pharmaceutical rules (pharmaceutical manufacturing) to prepare a preparation. The amount of active ingredient in these preparations is an appropriate capacity to obtain the indicated range.

The sterile composition for injection can be formulated according to the usual pharmaceutical preparation rules using a vehicle such as distilled water for injection.

Examples of aqueous solutions for injection include physiological saline and isotonic solutions containing glucose or other adjuvants (e.g., D-sorbitol, D-mannose, D-mannitol, sodium chloride). These aqueous solutions for injection may be used in combination with appropriate cosolvents, such as alcohols, specifically ethanol, polyols (e.g., propylene glycol, polyethylene glycol), and nonionic surfactants (e.g., polysorbate 80™, HCO-50).

Oily liquids include sesame oil and soybean oil. These oily liquids can be used in combination with benzyl benzoate or benzyl alcohol as cosolvents. They can also be mixed with buffers (e.g., phosphate buffer, sodium acetate buffer), analgesics (e.g., procaine hydrochloride), stabilizers (e.g., benzyl alcohol, phenol), and antioxidants. The prepared injection is typically filled into an appropriate ampoule.

The administration route is preferably parenteral administration, and specific examples include injection, nasal administration, transpulmonary administration, and transdermal administration. Examples of injections include those that can be administered systemically or locally via intravenous, intramuscular, intraperitoneal, and subcutaneous injections.

An appropriate administration method can be selected according to the patient's age and symptoms. The dosage of the pharmaceutical composition containing the antibody or the polynucleotide encoding the antibody can be selected, for example, within the range of 0.0001 mg to 1000 mg/kg body weight per dose. Alternatively, for example, the dosage can be selected within the range of 0.001 to 100,000 mg/person per patient, but is not limited to the above values. The dosage and administration method can vary according to the patient's weight, age, symptoms, etc., and can be appropriately selected by those skilled in the art.

The correspondence between the three-letter and one-letter amino acid symbols used in this specification is as follows.

Alanine: Ala(A)

Arginine: Arg(R)

Asparagine: Asn(N)

Aspartic acid: Asp(D)

Cysteine: Cys(C)

Glutamine: Gln(Q)

Glutamate: Glu(E)

Glycine: Gly(G)

Histidine: His(H)

Isoleucine: Ile(I)

Leucine: Leu(L)

Lysine: Lys(K)

Methionine: Met(M)

Phenylalanine: Phe(F)

Proline: Pro(P)

Serine: Ser(S)

Threonine: Thr(T)

Tryptophan: Trp(W)

Tyrosine: Tyr(Y)

Valine: Val(V)

It should be noted that all prior art documents cited in this specification are incorporated into this specification as reference.

Example

Hereinafter, the present invention will be specifically described with reference to Examples, but the present invention is not limited to these Examples.

[Example 1] Preparation of SR344 using affinity maturation technology to improve antigen binding ability through CDR modification

A CHO cell constant expression strain was constructed that consists of the soluble human IL-6R (hereinafter referred to as SR344) sequence consisting of amino acids 1 to 344 on the N-terminal side reported in J. Biochem. 108, 673-676 (1990) (Yamasaki et al., Science (1988) 241, 825-828 (GenBank #X12830)).

SR344 was purified from the culture supernatant obtained from SR344-expressing CHO cells by three types of column chromatography: Blue Sepharose 6 FF column chromatography, affinity chromatography on a column immobilized with an SR344-specific antibody, and gel filtration column chromatography.

The culture supernatant was directly applied to a Blue Sepharose 6 FF column (GE Healthcare Bio-Sciences) equilibrated with 20 mM Tris-HCl buffer (pH 8.0). Unadsorbed fractions were completely eluted with the same buffer. The column was then washed with the same buffer containing 300 mM KCl. The adsorbed protein was then eluted using a linear concentration gradient of KSCN from 0 M to 0.5 M in the same buffer containing 300 mM KCl. Fractions eluted with the KSCN concentration gradient were analyzed by Western blotting using an SR344-specific antibody, and fractions containing SR344 were collected.

The column immobilized with the SR344-specific antibody was pre-equilibrated with Tris-buffered saline (TBS). The SR344 fraction obtained in step 1 was ultrafiltered and concentrated using an Amicon Ultra-15 (MILLIPORE; molecular weight cutoff 10 kDa), diluted two-fold with TBS, and then added to the column. The column was washed with TBS, and the adsorbed protein was eluted with 100 mM glycine-HCl buffer (pH 2.5). 1 M Tris (pH 8.1) was added to the eluted fraction to return the pH to neutral. The resulting fractions were analyzed by SDS-PAGE, and the fraction containing SR344 was collected.

The fractions obtained in step 2 were concentrated using an Amicon Ultra-15 (molecular weight cutoff of 10 kDa) and then added to a Superdex 200 column (GE Healthcare Bio-Sciences) equilibrated with PBS. The fraction eluting as the main peak was used as the final pure product of SR344.

Establishment of BaF3 cell line expressing human gp130

In order to obtain a cell line showing IL-6-dependent growth, a BaF3 cell line expressing human gp130 was established as follows.

The full-length human gp130 cDNA (Hibi et al., Cell 1990; 63: 1149-1157 (GenBank # NM_002184)) was amplified by PCR and cloned into the expression vector pCOS2Zeo (which was created by removing the DHFR gene expression site from pCHOI (Hirata et al., FEBS Letters; 1994; 356: 244-248) and inserting the Zeocin resistance gene expression site) to construct pCOS2Zeo/gp130. The full-length human IL-6R cDNA was amplified by PCR and cloned into pcDNA3.1(+) (Invitrogen) to construct hIL-6R/pcDNA3.1(+).

10 μg of pCOS2Zeo/gp130 was mixed with BaF3 cells (0.8×10 ⁷ cells) suspended in PBS, and then pulsed at 0.33 kV and 950 μFD using a Gene Pulser (Bio-Rad). BaF3 cells transfected with the gene by electroporation were cultured overnight in RPMI 1640 medium (Invitrogen) supplemented with 0.2 ng/mL mouse interleukin-3 (Peprotech) and 10% fetal bovine serum (FBS; HyClone). Selection was performed by adding RPMI 1640 medium supplemented with 100 ng/mL human interleukin-6 (R&D Systems) and 100 ng/mL human interleukin-6 soluble receptor (R&D Systems) and 10% FBS. A BaF3 cell line expressing human gp130 (hereinafter referred to as BaF3/gp130) was established. Since BaF/gp130 proliferates in the presence of human interleukin-6 (R&D Systems) and SR344, it can be used to evaluate the growth inhibitory activity (ie, IL-6 receptor neutralizing activity) of anti-IL-6 receptor antibodies.

Construction of CDR modification library

First, the humanized PM-1 antibody (Cancer Res. 1993 Feb 15; 53(4):851-6) was converted into scFv. The VH and VL regions were amplified by PCR to produce a humanized PM-1HL scFv with a linker sequence GGGGSGGGGSGGGGS (SEQ ID NO: 106) between the VH and VL regions.

Using the prepared humanized PM-1 HL scFv DNA as a template, PCR was used to create a target library in which one amino acid in each CDR was designated as X, as well as a library in which only the hot spot sequence in the CDR was randomized. The target library in which only one amino acid in each CDR was designated as X was constructed by PCR using primers with the amino acid to be libraryized as the NNS. The remaining portions were prepared using standard PCR and ligated using assembly PCR. In this case, only one CDR was included in the library (see J. Mol. Biol. 1996; 256: 77-88). A library in which only the hot spot sequence was randomized was constructed using PCR using primers with all hot spot amino acids as NNS. In this case, a library containing only the VH hot spot and a library containing only the VL hot spot were constructed (see Nature Biotechnology 1999 June; 17: 568-572).

Using this library, a ribosome display library was constructed according to J. Immunological Methods 1999; 231: 119-135. To enable in vitro translation using an E. coli cell-free system, an SDA sequence (ribosome binding site) and a T7 promoter were added to the 5' end, and a partial gene3 sequence was used as a ribosome display linker, ligated to the 3' end using Sfil.

Obtaining high-affinity scFv via ribosome display

Panning was performed using ribosome display according to Nature Biotechnology 2000 Dec;18:1287-1292. The prepared SR344 was biotinylated with NHS-PEO4-Biotin (Pierce) and used as an antigen. To efficiently obtain high-affinity scFvs, off-rate selection was performed with reference to JBC 2004;279(18):18870-18877. The amount of biotinylated antigen was 1 nM, the amount of competing antigen was 1 μM, and the competition time in the fourth round was 10 O/N.

scFv: phagemid insertion, antigen binding activity, and sequence analysis

HL scFv was recovered by PCR using the DNA library obtained in round 4 as a template and specific primers. The fragment was digested with SfiI, and the resulting fragment was inserted into the phagemid vector pELBG lacI, which had also been digested with SfiI, and transformed into XL1-Blue (Stratagene). The resulting colonies were used for antigen binding activity evaluation and HL scFv sequence analysis using phage ELISA. Phage ELISA was performed using plates coated with 1 μg/mL SR344 according to J. Mol. Biol 1992;227:381-388. Clones that demonstrated SR344 binding activity were sequenced using specific primers.

Conversion from scFv to IgG, and expression and purification of IgG

IgG was expressed using an animal cell expression vector. Concentrated clones with confirmed mutation sites were amplified by PCR for their VH and VL, digested with XhoI/NheI and then digested with EcoRI, and the amplified DNA was inserted into an animal cell expression vector. The nucleotide sequence of each DNA fragment was determined using a BigDye Terminator Cycle Sequencing Kit (Applied Biosystems) using a DNA sequencer (ABI PRISM 3730xL DNA Sequencer or ABI PRISM 3700 DNA Sequencer (Applied Biosystems)) according to the method described in the appendix instructions.

Expression of IgG antibodies

The expression of the antibody was carried out by the following method. The HEK293H strain (Invitrogen) from human embryonic kidney cancer cells was suspended in DMEM medium (Invitrogen) containing 10% fetal bovine serum (Invitrogen), and 10 mL was seeded in each culture dish of an adherent cell culture dish (10 cm in diameter; CORNING) at a cell density of 5 to 6 × 10 ⁵ cells / mL. The culture dish was cultured in an incubator at 37 ° C and 5% CO ₂ for one day and night. After that, the culture medium was removed and 6.9 mL of CHO-S-SFM-II medium (Invitrogen) was added. The prepared plasmid DNA mixture (a total of 13.8 μg) was mixed with 20.7 μL of 1 μg / mL polyethyleneimine (Polysciences Inc.) and 690 μL of CHO-S-SFMII medium, allowed to stand at room temperature for 10 minutes, and then added to the cells in each culture dish and cultured in an incubator at 37 ° C and 5% CO ₂ for 4 to 5 hours. Afterwards, 6.9 mL of CHO-S-SFM-II (Invitrogen) medium was added and cultured in a _CO2 incubator for 3 days. The culture supernatant was recovered and then centrifuged at approximately 2000 g for 5 minutes at room temperature to remove cells. The cells were then sterilized by passing through a 0.22 μm filter ⁽ MILLEX®-GV (Millipore)). The ampoule was stored at 4°C until use.

Purification of IgG antibodies

To the obtained culture supernatant, add 50 μL rProtein A Sepharose ^™ Fast Flow (Amersham Biosciences) suspended in TBS and invert and mix at 4°C for more than 4 hours. The solution was transferred to a 0.22 μm filter cup Ultrafree ^(R) -MC (Millipore), washed three times with 500 μL TBS, and then the rProtein A Sepharose ^™ resin was suspended in 100 μL 50 mM sodium acetate aqueous solution (pH 3.3), allowed to stand for 2 minutes, and then the antibody was eluted. Immediately add 6.7 μL of 1.5 M Tris-HCl (pH 7.8) for neutralization. Elute twice to obtain 200 μL of purified antibody. 2 μL of the solution containing the antibody was supplied to a spectrophotometer ND-1000 (NanoDrop), or 50 μL of the solution containing the antibody was supplied to a spectrophotometer DU-600 (BECKMAN), and the absorbance at 280 nm was measured. The antibody concentration was calculated from the obtained values using the following formula.

Antibody concentration (mg/mL) = absorbance × dilution ratio ÷ 14.6 × 10

Evaluation of the neutralizing activity of human IL-6 receptor by IgG clones

IL-6 receptor neutralizing activity was evaluated using BaF3/gp130, which exhibits IL-6/IL-6 receptor-dependent proliferation. BaF3/gp130 was washed three times with RPMI1640 medium containing 10% FBS, then suspended in RPMI1640 medium containing 60 ng/mL human interleukin-6 (TORAY), 60 ng/mL recombinant soluble human IL-6 receptor (SR344), and 10% FBS to a density of 5×10 ⁴ cells/mL. 50 μL of the suspension was then injected into each well of a 96-well plate (CORNING). Next, the purified antibody was diluted in RPMI1640 medium containing 10% FBS, and 50 μL of the suspension was mixed into each well. After culturing for 3 days at 37°C and 5% _CO₂ , 20 μL/well of WST-8 reagent (Cell Counting Kit 8; Dojindo Chemical Laboratories, Ltd.) diluted 2-fold with PBS was added. Immediately thereafter, the absorbance at 450 nm (reference wavelength: 620 nm) was measured using a SUNRISE CLASSIC (TECAN). After culturing for 2 hours, the absorbance at 450 nm (reference wavelength: 620 nm) was measured again. The IL-6 receptor neutralizing activity was evaluated using the change in absorbance over 2 hours as an indicator.

As a result, several antibodies were obtained that exhibited activity higher than that of the humanized PM-1 antibody (wild-type: WT). The mutation sites of antibodies exhibiting activity higher than that of the WT are shown in Figure 4. For example, as shown in Figure 1, RD_6 exhibited approximately 50 times higher neutralizing activity than the WT at a 100% inhibitory concentration.

Affinity analysis of IgG clones using Biacore

For clones with higher activity than the wild type, Biacore T100 (BIACORE) was used to analyze the rate of antigen-antibody reaction. 1800RU~2600RU (resonance units) of rec-Protein A (ZYMED) (hereinafter referred to as Protein A) was immobilized on a sensor chip, various antibodies were bound to the chip, and the antigen was flowed through the chip as an analyte to measure the interaction between the antibody and the antigen. Recombinant human IL-6R sR (R&D Systems) (hereinafter referred to as rhIL-6sR) adjusted to various concentrations was used as an antigen. All measurements were performed at 25°C. The kinetic parameters, i.e., the binding rate constant _ka (1/Ms) and the dissociation rate constant _kd (1/s), were calculated from the sensorgrams obtained in the measurement, and _KD (M) was calculated based on the values. Each parameter was calculated using Biacore T100 evaluation software (BIACORE).

As a result, several antibodies were obtained with higher affinities than the humanized PM-1 antibody (wild-type: WT). For example, the sensorgrams for the wild-type (WT) and RD_6 are shown in Figures 2 and 3. Kinetic parameter analysis revealed that RD_6 exhibited approximately 50 times higher affinity than the WT (Table 1). Furthermore, antibodies were obtained that retained affinity several dozen times higher. The mutation sites that exhibited higher affinity than the WT are shown in Figure 4.

[Table 1]

[Example 2] Improving antigen binding ability by combining various CDR modifications

Mutation sites with high activity and affinity are fused to produce antibodies with higher activity and affinity.

Production, expression, and purification of modified antibodies

The selected sites were subjected to amino acid modifications for the preparation of modified antibodies. Specifically, the QuikChange site-directed mutagenesis kit (Stratagene) was used to introduce mutations into the H (WT) variable region (H (WT), SEQ ID NO: 107) and L (WT) chain variable region (L (WT), SEQ ID NO: 108) produced according to the method described in the appendix specification. The H chain antibody gene fragment confirmed to be the target humanized antibody variable region gene sequence was inserted into the plasmid with XhoI and NotI digestion, and then the plasmid with the L chain antibody gene fragment was digested with EcoRI, and then the reaction solution was subjected to 1% agarose gel electrophoresis. Using the QIAquick gel extraction kit (QIAGEN), the DNA fragment of the target size (about 400bp) was purified according to the method described in the appendix specification and eluted with 30 μL of sterile water. The antibody H chain gene fragment was then inserted into an animal cell expression vector to produce the target H chain expression vector. The same operation was performed to produce an L chain expression vector. Ligation reactions were performed using a Rapid DNA Ligation Kit (Roche Diagnostics) and transformed into Escherichia coli DH5α (Toyobo). The nucleotide sequence of each DNA fragment was determined using a BigDye Terminator Cycle Sequencing Kit (Applied Biosystems) on a DNA sequencer (ABI PRISM 3730xL DNA Sequencer or ABI PRISM 3700 DNA Sequencer (Applied Biosystems)) according to the methods described in the appendix. The prepared expression vectors were used for expression and purification according to the methods described in Example 1.

Evaluation of human IL-6 receptor neutralizing activity

The neutralizing activity of the purified antibodies was evaluated according to the method described in Example 1. Neutralizing activity was evaluated at a concentration of human interleukin-6 (TORAY) of 600 ng/mL. Several novel antibodies exhibiting activity higher than that of the WT were obtained, and the CDR sequences of these antibodies are shown in Figure 5. Among these, the neutralizing activity of an antibody using RDC_5H in the H chain and RDC_11L in the L chain (RDC_23), which exhibited high activity, is shown in Figure 6. It was found that RDC_23 had approximately 100-fold higher activity than the WT at a 100% inhibitory concentration. Improved neutralizing activity was observed not only in RDC_23, i.e., antibodies using RDC_5H for the H chain and RDC_11L for the L chain, but also in antibodies using RDC_2H, RDC_3H, RDC_4H, RDC_5H, RDC_6H, RDC_7H, RDC_8H, RDC_27H, RDC_28H, RDC_29H, RDC_30H, RDC_32H for the H chain and L (WT) for the L chain (formed respectively). Improved neutralizing activity was also confirmed in antibodies using H (WT) in the H chain and RDC_11L in the L chain (RDC_11). Combining mutation sites discovered through affinity maturation techniques can yield antibodies with higher activity. Furthermore, since antibodies combining these mutation sites exhibited improved neutralizing activity, it is believed that their affinity was also improved.

Affinity analysis using Biacore using Protein A

Thus, among the antibodies with improved neutralizing activity, for RDC_2, RDC_3, RDC_4, RDC_5, RDC_6, RDC_7, RDC_8, RDC_11, and RDC_23, Biacore T100 (BIACORE) was used to analyze the rate of antigen-antibody reaction. 4400RU to 5000RU of rec-protein A (ZYMED) was immobilized on a sensor chip by amine coupling, and various antibodies were bound to the chip. The antigen was then flowed on the chip as an analyte to measure the interaction between the antibody and the antigen. The antigen was adjusted to various concentrations of rhIL-6sR. All measurements were performed at 25°C. Kinetic parameters, i.e., the association rate constant _ka (1/Ms) and the dissociation rate constant _kd (1/s), were calculated from the sensorgrams obtained in the measurements, and _KD (M) was calculated based on these values. Each parameter was calculated using Biacore T100 evaluation software (BIACORE). The results showed that RDC_2, RDC_3, RDC_4, RDC_5, RDC_6, RDC_7, RDC_8, RDC_11, and RDC_23, which all contained combinations of mutations, had lower _K values than RD_28, which contained a single mutation and was also measured (Table 2). The sensorgram for RDC_23, which exhibited high affinity, is shown in Figure 7.

[Table 2]

This demonstrates that these antibodies exhibit enhanced affinity compared to the antibodies before the mutations were combined. Similar to neutralizing activity, combining mutations discovered through affinity maturation techniques can yield antibodies with even higher affinity. The amino acid sequences of mutants exhibiting activity or affinity exceeding WT are shown below (mutation sites derived from WT are underlined).

(HCDR2)

SEQ ID NO:45 YISYSGIT N YNPSLKS

(HCDR3)

SEQ ID NO:57 L LAR A TAMDY

SEQ ID NO:58 V LAR A TAMDY

SEQ ID NO:59 I LAR A TAMDY

SEQ ID NO:60 T LAR A TAMDY

SEQ ID NO:61 V LAR I TAMDY

SEQ ID NO:62 I LAR I TAMDY

SEQ ID NO:63 T LAR I TAMDY

SEQ ID NO:64 L LAR I TAMDY

(LCDR3)

SEQ ID NO:79 G QGN R LPYT

That is, by producing an antibody in which the 9th amino acid of HCDR2 is Asn, the 1st amino acid of HCDR3 is any one of Leu, Val, Ile, and Thr, the 5th amino acid of HCDR3 is Ala or Ile, the 1st amino acid of LCDR3 is Gly, and the 5th amino acid of LCDR3 is Arg, it is possible to produce an anti-IL-6 receptor antibody with significantly improved neutralizing activity and affinity compared to WT.

Affinity analysis using Biacore using Protein A/G

Using Biacore T100 (BIACORE), the speed of the antigen-antibody reaction of WT and RDC_23 was analyzed. Purified recombinant protein A/G (Pierce) (hereinafter referred to as protein A/G) was fixed on a sensor chip, various antibodies were bound to the chip, and the antigen was flowed on the chip in the form of an analyte to measure the interaction between the antibody and the antigen. Antigens were adjusted to various concentrations of rhIL-6sR (R&D Systems) and recombinant soluble IL-6 receptor (SR344 prepared in Example 1). The sugar chain structure of the rhIL-6sR produced by baculovirus-infected insect cells is a high mannose type. In contrast, the sugar chain structure of SR344 produced by CHO cells is considered to be a complex sugar chain and end-bound with sialic acid. In fact, the sugar chain structure of the soluble IL-6 receptor in the human body is a complex sugar chain with sialic acid attached to the end. Therefore, it is believed that the structure of SR344 is closer to the soluble IL-6 receptor in the human body. In this experiment, a comparative test between rhIL-6sR and SR344 was conducted.

Kinetic parameters, namely, the association rate constant _ka (1/Ms) and the dissociation rate constant _kd (1/s), were calculated from the sensorgrams obtained in the measurement, and _KD (M) was calculated based on these values. Each parameter was calculated using Biacore T100 evaluation software (BIACORE).

The sensor chip was made by immobilizing approximately 3000RU of protein A/G on CM5 (BIACORE) using the amine coupling method. Using the prepared sensor chip, the rate of interaction between antibodies (WT and RDC_23) bound to protein A/G and two soluble IL-6 receptors, rhIL-6sR and SR344, was analyzed. The running buffer used was HBS-EP+, and the flow rate was 20μL/min. Each antibody was prepared to approximately 100RU bound to protein A/G. RhIL-6sR used as the analyte was prepared using HBS-EP+ at 0, 0.156, 0.313, and 0.625μg/mL. SR344 was prepared at 0, 0.0654, 0.131, and 0.261μg/mL. The assay first involved binding the target antibodies WT and RDC_23 to Protein A/G. The analyte solution was then allowed to react for 3 minutes. The assay then switched to HBS-EP+ (BIACORE) and the dissociation phase was measured for 10 minutes. Following the dissociation phase, the sensor chip was washed with 10 μL of 10 mM glycine-HCl (pH 1.5) to regenerate. This binding, dissociation, and regeneration cycle constituted one analysis cycle. All experiments were performed at 37°C.

WT and RDC_23 were measured using the above-described cycles, and the resulting sensorgrams for rhIL-6sR and SR344, two soluble IL-6 receptors, are shown in Figures 8, 9, 10, and 11. The resulting sensorgrams were analyzed using Biacore T100 evaluation software, a data analysis software specifically for Biacore (Table 3). The results show that, in a comparison of rhIL-6sR and SR344, both WT and RDC_23 exhibited a 2- to 3-fold weaker affinity than that obtained with SR344. Compared to WT, RDC_23 exhibited approximately 40- to 60-fold higher affinity for both rhIL-6sR and SR344. Using a combination of CDR modifications derived through affinity maturation techniques, RDC_23 exhibited significantly stronger affinity for SR344, which is believed to be structurally similar to the soluble IL-6 receptor in humans, compared to WT. In the following examples, the analysis of the rate of antigen-antibody reaction using SR344 and protein A/G was performed at 37°C.

[Table 3]

[Example 3] Improvement of plasma retention and reduction of immunogenicity risk by modification of CDRs and framework Development of H53/L28

Cancer Res. 1993 Feb 15; 53(4): 851-6 describes the following modifications to humanized mouse PM-1 antibody (hereinafter referred to as wild type or WT; H chain WT referred to as H(WT); L chain WT referred to as L(WT)) to improve plasma retention, reduce the risk of immunogenicity, and enhance stability. To improve plasma retention, modifications were introduced into the H and L chain variable region sequences of the WT to lower the isoelectric point.

Preparation of a three-dimensional structural model of the humanized PM-1 antibody

First, to identify the amino acid residues exposed on the variable region surface of the humanized PM-1 antibody (H(WT)/L(WT)), a humanized mouse PM-1 antibody Fv region model was constructed by homology modeling using MOE software (Chemical Computing Group Inc.).

Selection of modification sites for lowering the isoelectric point of humanized PM-1 antibody

Through detailed analysis of the prepared model, it was found that H16, H43, H81, H105, L18, L45, L79, and L107 (Kabat numbering; Kabat EA et al., 1991, Sequences of Proteins of Immunological Interest (target immune protein sequences), NIH) among the surface-exposed amino acids in the FR sequence, and H31, H64, H65, L24, L27, L53, and L55 in the CDR sequence were candidate sites for lowering the isoelectric point without reducing activity and stability.

Removal of residual mouse sequences from humanized PM-1 antibody

The humanized PM-1 antibody is an antibody sequence obtained by humanizing the mouse PM-1 antibody (Cancer Res. 1993 Feb 15; 53(4): 851-6). The H chain of the humanized PM-1 antibody is obtained by grafting the CDRs onto a new framework, which is a human antibody variable region. However, H27, H28, H29, H30, and H71 of the H chain are directly derived from mouse sequences to maintain activity. Considering the risk of immunogenicity, it is believed that the less mouse sequences, the better. Therefore, the sequence used to convert H27, H28, H29, and H30 into human sequences was explored.

Selection of modification sites for improving the stability of humanized PM-1 antibody

The present inventors believe that stability can be improved by substituting serine at H65 with glycine in the variable region of the humanized PM-1 antibody (H(WT)/L(WT)) (stabilization of the turn structure; stabilization achieved by converting HCDR2 to a consensus sequence), methionine at H69 with isoleucine (stabilization of the hydrophobic core structure), leucine at H70 with serine (stabilization achieved by hydrophilizing surface-exposed residues), threonine at H58 with asparagine (stabilization achieved by converting HCDR2 to a consensus sequence), threonine at L93 with serine (stabilization achieved by hydrophilizing surface-exposed residues), and serine at H107 with isoleucine (stabilization of the β-sheet), and believe that these modifications are candidates for improving stability.

Removal of in silico predicted T cell epitopes of humanized PM-1 antibody

First, the variable region of the humanized PM-1 antibody (H(WT)/L(WT)) was analyzed using TEPITOPE (Methods 2004 Dec;34(4):468-75). The results showed that multiple HLA-binding T-cell epitopes are present in the L-chain CDR2. Therefore, TEPITOPE analysis was conducted to investigate modifications that could reduce the immunogenicity risk of the L-chain CDR2 without compromising stability, binding activity, or neutralizing activity. The results showed that substituting threonine at L51 of the L-chain CDR2 with glycine could eliminate HLA-binding T-cell epitopes without compromising stability, binding activity, or neutralizing activity.

Selection of each framework sequence

Human antibody amino acid sequence data was obtained from the generally open Kabat database (ftp://ftp.ebi.ac.uk/pub/databases/kabat/) and the IMGT database (http://imgt.cines.fr/). Using the database constructed therefrom, homology can be retrieved by distinguishing each framework. When selecting a human framework, the human framework sequence with the above-mentioned modifications was studied using the database from the perspectives of lowering the isoelectric point, removing residual mouse sequences, and improving stability. As shown in the results, by forming the following sequences for each framework of the modified antibody H53/L28, the above-mentioned items can be met without reducing binding activity and neutralization activity. Source is the source of the human sequence, and the amino acid residues in the underlined portion of the sequence are modified amino acids introduced compared to WT.

[Table 4]

Since the FR3 of H53 described above contains non-human sequences, it is desirable to further reduce the risk of immunogenicity. As a modification to reduce the risk of immunogenicity, it is believed that a sequence in which Ala at H89 is substituted with Val can be formed (SEQ ID NO: 127). Furthermore, since Arg at position H71 in the FR3 of H53 is important for binding activity (Cancer Res. 1993 Feb 15; 53(4): 851-6), it is believed that by using the FR3 sequence of the human VH1 subclass (SEQ ID NO: 128) or the FR3 sequence of the human VH3 subclass (SEQ ID NO: 129) with Arg at position H71, it is possible to produce an anti-human IL-6 receptor antibody in which both the H chain and L chain frameworks are completely human sequences.

Selection of each CDR sequence

When selecting CDR sequences, first, the considerations were to lower the isoelectric point, improve stability, and remove T cell epitopes without reducing binding and neutralizing activity. The CDR sequences of H53/L28 were selected as follows.

[Table 5]

H53 sequence CDR1 CDR2 CDR3 SLARTTAMDY L28 sequence CDR1 CDR2 CDR3

Preparation, expression, and purification of expression vectors for modified antibodies

The modified antibody expression vectors were prepared, expressed, and purified according to the methods described in Example 1. Modifications were sequentially introduced into the H (WT) and L (WT) mutation vectors of the humanized mouse PM-1 antibody to create the selected framework and CDR sequences. The resulting animal cell expression vector encoding H53/L28 (antibody amino acid sequences: H53, SEQ ID NO: 104; L28, SEQ ID NO: 105) with the selected framework and CDR sequences was used to express and purify H53/L28 and used in the following evaluations.

Evaluation of the isoelectric point of the modified antibody H53/L28 by isoelectric point electrophoresis

To evaluate the changes in the isoelectric point of the full-length antibody caused by the amino acid modifications in the variable regions, WT and modified antibodies H53/L28 were analyzed by isoelectric electrophoresis. Isoelectric electrophoresis was performed as follows. Using a Phastsystems Cassette (Amersham Biosciences), a Phast-Gel Dry IEF (Amersham Biosciences) gel was swollen in the following swelling solution for approximately 30 minutes.

Milli-Q water 1.5 mL

Pharmalyte 5-8 (Amersham Biosciences) for IEF 50 μL

Pharmalyte 8-10.5 (Amersham Biosciences) for IEF 50 μL

Using the swollen gel, electrophoresis was performed using Phast Systems (Amersham Biosciences) according to the following procedure: The sample was added to the gel in step 2. For pI markers, a pI calibration kit (Amersham Biosciences) was used.

After electrophoresis, the gel was fixed with 20% TCA and then silver stained using a silver staining kit and protein (made by Amersham Biosciences) according to the instructions included in the kit. After staining, the isoelectric point of the sample (full-length antibody) was calculated from the known isoelectric point labeled with pI. As a result, the isoelectric point of WT was about 9.3, and the isoelectric point of the modified antibody H53/L28 was about 6.5-6.7. By making amino acid substitutions in WT, H53/L28 with an isoelectric point lowered by about 2.7 was obtained. When the theoretical isoelectric point of the H53/L28 variable region (VH, VL sequence) was calculated by GENETYX (GENETYX CORPORATION), the theoretical isoelectric point was 4.52. Since the theoretical isoelectric point of WT is 9.20, H53/L28 with a theoretical isoelectric point of the variable region lowered by about 4.7 was obtained by making amino acid substitutions in WT.

Evaluation of the human IL-6 receptor neutralizing activity of H53/L28

WT and H53/L28 were evaluated according to the method described in Example 1. The results are shown in Figure 12. The results demonstrate that the modified antibody H53/L28 exhibits several-fold increased neutralization activity against BaF/gp130 compared to WT. This indicates that H53/L28 exhibits enhanced neutralization activity compared to WT without lowering its isoelectric point.

Analysis of the affinity of H53/L28 for human IL-6 receptor using Biacore

The affinity of WT and H53/L28 for the human IL-6 receptor was evaluated using a Biacore T100 (BIACORE) kinetic analysis. Purified recombinant Protein A/G (Pierce) (hereinafter referred to as Protein A/G) was immobilized on a sensor chip. Various antibodies were bound to the chip, and the antigen was flowed across the chip as an analyte to measure the antibody-antigen interaction. The antigen used was recombinant soluble IL-6 receptor (SR344) at various concentrations. The assay conditions were the same as in Example 2.

The resulting sensorgrams for WT and H53/L28 are shown in Figure 13. Velocity analysis was performed using Biacore T100 evaluation software, a data analysis software specifically designed for Biacore. The results are shown in Table 6. The results show that the _KD of H53/L28 decreased approximately 6-fold compared to WT, while the affinity increased approximately 6-fold. In other words, H53/L28 was able to increase affinity by approximately 6-fold without lowering the isoelectric point compared to WT. Detailed research results suggest that the amino acid mutation that contributes to increased affinity is the substitution of threonine at L51 for glycine. In other words, it is believed that substitution of threonine at L51 for glycine can improve affinity.

[Table 6]

Prediction of T cell epitopes in H53/L28 using TEPITOPE

TEPITOPE analysis of H53/L28 (Methods. 2004 Dec; 34(4): 468-75) revealed that the number of peptides potentially binding to HLA in H53/L28 was significantly reduced compared to WT, suggesting that the risk of immunogenicity in humans is reduced.

[Example 4] Evaluation of plasma retention of H53/L28

Evaluation of the kinetics of the modified antibody H53/L28 in normal mouse plasma

To evaluate the plasma retention of the modified antibody H53/L28 with a lowered isoelectric point, the kinetics of WT and modified antibody H53/L28 in normal mouse plasma were compared.

WT and H53/L28 were administered intravenously and subcutaneously to mice (C57BL/6J; Charles River, Japan) at a single dose of 1 mg/kg, and blood was collected before and 15 minutes, 2 hours, 8 hours, 1 day, 2 days, 5 days, 7 days, 14 days, 21 days, and 28 days after administration. Among them, blood was collected only from the intravenous administration group 15 minutes after administration. The collected blood was immediately centrifuged at 15,000 rpm for 15 minutes at 4°C to obtain plasma. The separated plasma was kept in a cold storage set to below -20°C before the measurement was performed.

The concentration in mouse plasma was determined by ELISA. First, recombinant human IL-6sR (R&D Systems) was biotinylated using the EZ-LinkTM Sulfo-NFS-biotinylation kit (PIERCE). The biotinylated human-sIL-6R was injected into a plate coated with Reacti-Bind high binding energy streptavidin (HBC) (PIERCE) and allowed to stand at room temperature for more than 1 hour to prepare a human-sIL-6R-immobilized plate. Standard curve samples with plasma concentrations of 3.2, 1.6, 0.8, 0.4, 0.2, 0.1, and 0.05 μg/mL and mouse plasma measurement samples were prepared and injected into the human-sIL-6R-immobilized plate, respectively, and allowed to stand at room temperature for 1 hour. Anti-human IgG-AP (Sigma) was then reacted with the antibody, and a colorimetric reaction was performed using the BluePhos Microwell Phosphatase Substrate System (Kirkegaard & Perry Laboratories) as a substrate. The absorbance at 650 nm was measured using a microplate reader. Mouse plasma concentrations were calculated from the absorbance of the standard curve using SOFTmax PRO (Molecular Devices) analysis software. Changes in plasma concentrations after intravenous administration of WT and H53/L28 are shown in Figure 14 , and changes in plasma concentrations after subcutaneous administration are shown in Figure 15 .

The obtained plasma concentration change data were subjected to model-independent analysis using the pharmacokinetic analysis software WinNonlin (manufactured by Pharsight) to calculate pharmacokinetic parameters (AUC, clearance (CL), and half-life (T1/2)). T1/2 was calculated from the plasma concentration at the last three points or the final phase automatically set by WinNonlin. BA was calculated from the ratio of the AUC after subcutaneous administration to the AUC after intravenous administration. The obtained pharmacokinetic parameters are shown in Table 7.

[Table 7]

After intravenous administration, the plasma half-life (T1/2) of H53/L28 was extended to approximately 1.3 times that of WT, and the clearance rate was reduced by approximately 1.7 times. After subcutaneous administration, the T1/2 of H53/L28 was extended to approximately 2 times that of WT, and the clearance rate was reduced by approximately 2.1 times. Thus, by lowering the isoelectric point of WT, the plasma retention of H53/L28 can be significantly improved.

Compared to the humanized PM-1 antibody (WT), H53/L28 is a humanized anti-IL-6 receptor antibody with enhanced binding and neutralizing activity, reduced immunogenicity risk, and significantly improved plasma retention. Therefore, the modifications applied to H53/L28 are considered extremely useful in drug development.

[Example 5] Preparation of PF1 Antibody

Preparation of expression vector and mutation introduction vector for humanized PM-1 antibody

Two H chain and two L chain CDR mutations, discovered in Example 2 to improve affinity for RDC_23, were introduced into H53/L28 prepared in Example 4, for a total of four CDR mutations. The H chain into which the RDC_23 mutations were introduced was designated PF1_H, and the L chain into which the RDC_23 mutations were introduced was designated PF1_L. The modified antibodies were prepared, expressed, and purified according to the methods described in Example 1. The amino acid sequence of PF1_H is shown in SEQ ID NO: 22, and the amino acid sequence of PF1_L is shown in SEQ ID NO: 23.

Evaluation of human IL-6 receptor neutralizing activity

The neutralizing activity of the purified PF1 antibody was evaluated according to the method described in Example 1. Neutralizing activity was assessed at a concentration of 600 ng/mL for human interleukin-6 (TORAY). The neutralizing activity of WT and PF1 is shown in Figure 16 . PF1 exhibits approximately 100- to 1000-fold greater activity than WT at a 100% inhibitory concentration.

Biacore analysis of the affinity of PF1 antibody to human IL-6 receptor

This determination was carried out under the same conditions as in Example 2. HBS-EP+ was used as the running buffer at a flow rate of 20 μL/min. Each antibody was prepared to approximately 100 RU and bound to protein A/G. HBS-EP+ was used for SR344 used as the analyte and adjusted to 0, 0.065, 0.131, and 0.261 μg/mL. During the determination, the antibody solution was first bound to protein A/G, and the analyte solution was allowed to react with it for 3 minutes, after which it was switched to HBS-EP+ and the dissociation phase was measured for 10 minutes or 15 minutes. After the determination of the dissociation phase was completed, the sensor chip was regenerated by washing with 10 μL of 10 mM glycine-HCl (pH 1.5). This binding, dissociation, and regeneration constitute one analysis cycle. Various antibodies were measured according to this cycle.

The resulting sensorgram for PF1 is shown in Figure 17. The resulting sensorgram was analyzed using Biacore T100 Evaluation Software, a data analysis software specifically designed for Biacore. The results, along with those for WT and H53/L28, are shown in Table 8. The results demonstrate that PF1 exhibits an approximately 150-fold increase in affinity compared to WT. By combining RDC_23, a high-affinity binding protein obtained through affinity maturation, with H53/L28, which exhibits enhanced plasma retention and affinity, PF1 exhibits a synergistic effect, exhibiting a higher affinity than either RDC_23 or H53/L28.

[Table 8]

Evaluation of the thermal stability of PF1 antibody using differential scanning calorimetry (DSC)

To evaluate the thermal stability of the PF1 antibody, differential scanning calorimetry (DSC) was used to assess the thermal denaturation intermediate temperature (Tm). The purified WT and PF1 antibodies were dialyzed against a solution of 20 mM sodium acetate, 150 mM NaCl, pH 6.0 (EasySEP, TOMY). DSC measurements were performed at a protein concentration of approximately 0.1 mg/mL, within a temperature range of 40°C to 100°C, at a heating rate of 1°C/minute. The results showed that the Tm value of the WT Fab portion was approximately 94°C, while that of the PF1 Fab portion was 91°C. The Tm value of the Fab portion of a typical IgG1 antibody molecule is in the range of approximately 60°C to 85°C (Biochem.Biophys.Res.Commun.2007 Apr 13;355(3):751-7;Mol Immunol.2007 Apr;44(11):3049-60), indicating that the resulting PF1 antibody has an extremely high thermal stability compared to a typical IgG1 molecule.

Evaluation of the stability of PF1 antibody at high concentrations

The stability of the PF1 antibody in a high-concentration formulation was evaluated. Purified WT and PF1 antibodies were dialyzed (EasySEP, TOMY) against a 20 mM histidyl chloride, 150 mM NaCl, pH 6.5 solution, then concentrated using an ultrafiltration membrane. Stability testing at high concentrations was performed using the following conditions.

Antibodies: WT and PF1

Buffer: 20 mM histidyl chloride, 150 mM NaCl, pH 6.0

Concentration: 145 mg/mL

Storage temperature and storage time: 2 weeks at 25°C, 4 weeks at 25°C, or 7 weeks at 25°C

Aggregate evaluation method:

System: Waters Alliance

Column: G3000SWxl (TOSOH)

Mobile phase: 50 mM sodium phosphate, 300 mM KCl, pH 7.0

Flow rate, wavelength: 0.5mL/min, 220nm

The samples were diluted 100-fold before analysis

The above-mentioned gel filtration chromatography method was used to evaluate the aggregate content in the original preparation (fresh preparation) and in preparations stored under various conditions. The change (increase) in the aggregate content in the original preparation is shown in Figure 18. The results show that both WT and PF1 showed very high stability. After 7 weeks of storage at 25°C, the aggregate content increased by approximately 0.7% in WT and 0.3% in PF1. After 1 month of storage at 25°C, the aggregate content increased by approximately 0.4% in WT and 0.17% in PF1. This shows that PF1 exhibits extremely high stability even at particularly high concentrations. WO 2003/039485 provides stability data at 25°C for a 100 mg/mL formulation of daclizumab, a marketed high-concentration IgG formulation. However, the increase in aggregates in the 100 mg/mL formulation of daclizumab after storage at 25°C for one month was approximately 0.3%. Even compared to daclizumab, PF1 exhibits extremely excellent stability at high concentrations. While the increase in aggregates is a significant issue in the development of high-concentration solution formulations as pharmaceuticals, the increase in aggregates for the PF1 antibody at high concentrations is minimal.

PF1 is a molecule modified from WT to achieve the following goals: enhanced antigen-binding ability, improved plasma retention by lowering the isoelectric point, reduced immunogenicity risk by removing residual mouse sequences and T-cell epitopes, and improved stability. PF1 has been shown to be significantly more stable than WT in high-concentration formulations of 100 mg/mL and above. Using this molecule, it is possible to provide a stable and convenient high-concentration subcutaneous formulation.

[Example 6] PK/PD study of PF1 antibody using human IL-6 receptor transgenic mice

In vivo kinetic studies using human IL-6 receptor transgenic mice

The kinetics of WT and PF1 prepared in Example 5 in human IL-6 receptor transgenic mice (hIL-6R tg mice; Proc. Natl. Acad. Sci. USA. 1995 May 23; 92(11): 4862-6) and the neutralization activity of human soluble IL-6 receptor in vivo were evaluated. WT and PF1 were administered intravenously to hIL-6Rtg mice at a single dose of 10 mg/kg, and blood was collected before administration and 15 minutes, 2 hours, 4 hours, 8 hours, 1 day, 2 days, 4 days, and 7 days after administration. The collected blood was immediately centrifuged at 15,000 rpm at 4°C for 15 minutes to obtain plasma. The separated plasma was stored in a refrigerator set at -20°C or below until the measurement was performed.

The concentration in mouse plasma was determined by ELISA. A standard curve sample with a plasma concentration of 6.4, 3.2, 1.6, 0.8, 0.4, 0.2, and 0.1 μg/mL was prepared. The standard curve sample and the mouse plasma assay sample were injected into a microplate (immunoplate) (Nunc-Immuno Plate, MaxiSorp (Nalgen International)) fixed with anti-human IgG (γ-chain specific) F(ab')2 (Sigma), and allowed to stand at room temperature for 1 hour. The plate was then reacted with goat anti-human IgG-BIOT (Southern Biotechnology Associates) and streptavidin alkaline phosphatase conjugate (Roche Diagnostics) in sequence, and a color reaction was performed using the BluePhos Microwell phosphatase substrate system (Kirkegaard & Perry Laboratories) as a substrate. The absorbance at 650 nm was measured using a microplate reader. Mouse plasma concentrations were calculated from the absorbance of the standard curve using the analysis software SOFTmax PRO (Molecular Devices). Changes in plasma concentrations of WT and PF1 are shown in Figure 19 . PF1 plasma concentrations were approximately fivefold higher than those of WT four days after administration, demonstrating that PF1 has enhanced plasma retention in human IL-6 receptor transgenic mice compared to WT.

Human IL-6 receptor transgenic mice are known to produce human soluble IL-6 receptor in their plasma. Therefore, by administering anti-human IL-6 receptor antibodies to human IL-6 receptor transgenic mice, the neutralization effect of human soluble IL-6 receptor present in plasma can be evaluated.

To evaluate the extent of neutralization of human soluble IL-6 receptor by administration of WT or PF1, the concentration of unbound human soluble IL-6 receptor in mouse plasma was measured. 6 μL of mouse plasma was diluted 2-fold with dilution buffer containing BSA and added to an appropriate amount of rProtein A Sepharose Fast Flow Resin (GE Healthcare) dried in a 0.22 μm filter cup (Millipore). All IgG antibodies present in the plasma (mouse IgG, anti-human IL-6 receptor antibody, and anti-human IL-6 receptor antibody-human soluble IL-6 receptor complex) were adsorbed on Protein A. The solution was then spun down in a high-speed centrifuge, and the flow-through solution was recovered. Since the flow-through solution does not contain the anti-human IL-6 receptor antibody-human soluble IL-6 receptor complex bound to Protein A, the concentration of unbound soluble IL-6 receptor can be determined by measuring the soluble IL-6 receptor concentration in the flow-through solution. Soluble IL-6 receptor concentrations were measured using Quantikine human IL-6 sR (R&D Systems). WT and PF1 were administered to mice, and unbound soluble IL-6 receptor concentrations were measured 4, 8, 24, 48, 96, and 168 hours later according to the instructions in the appendix.

The results are shown in Figure 20. Four to eight hours after a single intravenous dose of 10 mg/kg of WT and PF1, the concentration of unbound soluble IL-6 receptor was below 10 ng/mL in both WT and PF1, confirming neutralization of the human soluble IL-6 receptor. However, while the concentration of unbound soluble IL-6 receptor was approximately 500 ng/mL 24 hours after WT administration, the concentration of unbound soluble IL-6 receptor after PF1 administration was below 10 ng/mL, demonstrating that PF1 is able to sustain neutralization of the human soluble IL-6 receptor compared to WT.

PF1 is a combination of RDC_23, discovered through affinity maturation, and H53/L28, which has improved plasma retention. It is believed to exhibit prolonged plasma retention and high neutralizing activity in vivo. In fact, PF1 exhibited a neutralizing effect and sustained plasma concentrations compared to WT in human IL-6 receptor transgenic mice expressing the human soluble IL-6 receptor.

PF1 exhibits superior stability and immunogenicity risk compared to WT (humanized PM-1 antibody) in high-concentration formulations. Furthermore, it exhibits excellent IL-6 receptor neutralization and plasma retention even in human IL-6 receptor transgenic mice. Therefore, modifications applied to PF1 are considered extremely useful in drug development.

[Example 7] Improving the stability of IgG2 and IgG4 under acidic conditions

Preparation and expression of IgG2 and IgG4 humanized IL-6 receptor antibody expression vectors

Although the constant region of the humanized PM-1 antibody (Cancer Res. 1993 Feb 15; 53(4): 851-6) is of the IgG1 isotype, to reduce its binding activity to Fcγ receptors, molecules in which the constant region was substituted with IgG2 (WT-IgG2, SEQ ID NO: 109) and molecules in which the constant region was substituted with IgG4 (Mol. Immunol. 1993 Jan; 30(1): 105-8) (WT-IgG4, SEQ ID NO: 110) were prepared. Animal cell expression vectors were used for IgG expression. The constant region portion of the humanized PM-1 antibody (IgG1) used in Example 1 was digested with NheI/NotI, and then an expression vector was constructed in which the constant region was substituted with IgG2 or IgG4 by ligation. The nucleotide sequence of each DNA fragment was determined using the BigDye Terminator Cycle Sequencing Kit (Applied Biosystems) and a DNA sequencer (ABI PRISM 3730xL DNA Sequencer or ABI PRISM 3700 DNA Sequencer (Applied Biosystems)) according to the methods described in the appendix instructions. WT-IgG1, WT-IgG2, and WT-IgG4 were expressed as described in Example 1, using WT as the L chain.

(1) Humanized PM-1 antibody (WT-IgG1) H chain: SEQ ID NO: 15 (amino acid sequence)

(2) WT-IgG2 H chain: SEQ ID NO: 109 (amino acid sequence)

(3) WT-IgG4 H chain: SEQ ID NO: 110 (amino acid sequence)

Purification of WT-IgG1, WT-IgG2, and WT-IgG4 from Protein A by elution with hydrochloric acid

To the obtained culture supernatant, 50 μL of rProteinA Sepharose ^™ FastFlow (Amersham Biosciences) suspended in TBS was added and inverted and mixed at 4°C for more than 4 hours. The solution was transferred to a 0.22 μm filter cup Ultrafree ^(R) -MC (Millipore) and washed three times with 500 μL of TBS. The rProteinA Sepharose ^™ resin was then suspended in 100 μL of 10 mM HCl, 150 mM NaCl (pH 2.0), allowed to stand for 2 minutes, and then the antibody was eluted (hydrochloric acid elution method). Immediately, 6.7 μL of 1.5 M Tris-HCl (pH 7.8) was added for neutralization. Elution was performed twice to obtain 200 μL of purified antibody.

Gel filtration chromatography analysis of WT-IgG1, WT-IgG2, and WT-IgG4 purified by hydrochloric acid elution

In order to evaluate the aggregate content of the pure product obtained by the hydrochloric acid elution method, gel filtration chromatography analysis was performed.

Aggregate evaluation method:

System: Waters Alliance

Column: G3000SWxl (TOSOH)

Mobile phase: 50 mM sodium phosphate, 300 mM KCl, pH 7.0

Flow rate, wavelength: 0.5mL/min, 220nm

The results are shown in Figure 21. The aggregate content of purified WT-IgG1 was approximately 2%, while that of purified WT-IgG2 and WT-IgG4 was approximately 25%. This suggests that IgG1 is stable to the acid during hydrochloric acid elution, while IgG2 and IgG4 are unstable, denaturing and aggregating. This suggests that IgG2 and IgG4 are less stable under acidic conditions than IgG1. Protein A is often used to purify IgG molecules, and elution from Protein A is performed under acidic conditions. Furthermore, viral inactivation is essential for the development of IgG molecules as pharmaceuticals, and this inactivation is typically performed under acidic conditions. This suggests that, while IgG molecules are expected to be highly stable under acidic conditions, IgG2 and IgG4 are less stable than IgG1 under acidic conditions, demonstrating for the first time that denaturation and aggregation under acidic conditions are a problem for IgG2 and IgG4 molecules when developing them as pharmaceuticals. While it is considered desirable to address the denaturation and aggregation issues of IgG2 and IgG4 molecules when developing them as pharmaceuticals, no method has been reported to date that addresses these issues through amino acid substitution.

Preparation and evaluation of CH3 domain modifications of WT-IgG2 and WT-IgG4

Since the stability of IgG2 and IgG4 molecules under acidic conditions is poorer than that of IgG1, modifications in which the stability of IgG2 and IgG4 molecules under acidic conditions is improved are studied. According to the constant region model of IgG2 and IgG4 molecules, it is believed that the main cause of instability under acidic conditions is the instability of the CH3/CH3 interface in the CH3 domain. Various studies have been conducted, and the results show that the methionine at EU numbering position 397 in IgG2 and the arginine at EU numbering position 409 in IgG4 destabilize the CH3/CH3 interface of IgG2 and IgG4, respectively. Therefore, an antibody (IgG2-M397V, SEQ ID NO: 111 (amino acid sequence)) in which the methionine at EU numbering position 397 of IgG2 is changed to valine and an antibody (IgG4-R409K, SEQ ID NO: 112 (amino acid sequence)) in which the arginine at EU numbering position 409 of IgG4 is changed to lysine were prepared.

The expression vector for the target antibody was prepared, expressed, and purified using the hydrochloric acid elution method described above. Gel filtration chromatography was performed to evaluate the aggregate content of the purified protein A obtained by the hydrochloric acid elution method.

Aggregate evaluation method:

System: Waters Alliance

Column: G3000SWxl (TOSOH)

Mobile phase: 50 mM sodium phosphate, 300 mM KCl, pH 7.0

Flow rate, wavelength: 0.5mL/min, 220nm

The results are shown in Figure 21. The aggregate content of purified WT-IgG1 was approximately 2%, while that of purified WT-IgG2 and WT-IgG4 was approximately 25%. In contrast, the aggregate content of CH3 domain modifications IgG2-M397V and IgG4-R409K was approximately 2%, comparable to that of IgG1. Studies have shown that the stability of IgG2 and IgG4 antibodies under acidic conditions can be improved by replacing the methionine at EU position 397 of IgG2 with valine, or by replacing the arginine at EU position 409 of IgG4 with lysine. Furthermore, the thermal denaturation intermediate temperatures of WT-IgG2, WT-IgG4, IgG2-M397V, and IgG4-R409K were measured using the same method as in Example 5. The results showed that the Tm values of IgG2-M397V and IgG4-R409K, each containing a modified CH3 domain, were higher than those of WT-IgG2 and WT-IgG4. This revealed that both IgG2-M397V and IgG4-R409K were superior in thermal stability compared to WT-IgG2 and WT-IgG4.

Because IgG2 and IgG4 are exposed to acidic conditions during the purification and viral inactivation steps using Protein A, denaturation and aggregation during these steps become problematic. However, using IgG2-M397V and IgG4-R409K as the constant region sequences for IgG2 and IgG4 can address these issues. These modifications are therefore extremely useful in developing IgG2 and IgG4 antibodies as pharmaceuticals. IgG2-M397V and IgG4-R409K are also useful given their excellent thermal stability.

[Example 8] Improving the heterogeneity of disulfide bonds from IgG2

Purification of WT-IgG1, WT-IgG2, and WT-IgG4 from Protein A by elution with acetic acid

To the culture supernatant obtained in Example 7, 50 μL of rProteinA Sepharose ^™ Fast Flow (Amersham Biosciences) suspended in TBS was added and mixed inverted at 4°C for more than 4 hours. The solution was transferred to a 0.22 μm filter cup Ultrafree ^(R) -MC (Millipore) and washed three times with 500 μL of TBS. The rProtein A Sepharose ^™ resin was then suspended in 100 μL of 50 mM sodium acetate aqueous solution (pH 3.3), allowed to stand for 2 minutes, and then the antibody was eluted. Immediately, 6.7 μL of 1.5 M Tris-HCl (pH 7.8) was added for neutralization. Elution was performed twice to obtain 200 μL of purified antibody.

Cation exchange chromatography (IEC) analysis of WT-IgG1, WT-IgG2, and WT-IgG4

In order to evaluate the homogeneity of the purified WT-IgG1, WT-IgG2, and WT-IgG4, analysis was performed by cation exchange chromatography.

IEC evaluation method:

System: Waters Alliance

Column: ProPac WCX-10 (Dionex)

Mobile phase A: 25 mM MES-NaOH, pH 6.1

B: 25 mM MES-NaOH, 250 mM sodium acetate, pH 6.1

Flow rate, wavelength: 0.5mL/min, 280nm

Gradient B: 50%-75% (75 minutes) when analyzing WT-IgG1

B: 30%-55% (75 minutes) when analyzing WT-IgG2 and WT-IgG4

The results are shown in Figure 22. The results indicate that, in ion exchange analysis, WT-IgG1 and WT-IgG4 exhibit single peaks, while WT-IgG2 exhibits multiple peaks, indicating that IgG2 molecules exhibit greater heterogeneity compared to IgG1 and IgG4. In fact, heterogeneity (inhomogeneity) of IgG2 isotypes due to disulfide bonds in the hinge region has been reported (Chu GC, Chelius D, Xiao G, Khor HK, Coulibaly S, Bondarenko PV. Accumulation of Succinimide in a Recombinant Monoclonal Antibody in Mildly Acidic Buffers Under Elevated Temperatures. Pharm Res. 2007 Mar 24; 24(6): 1145-56), and the IgG2 miscellaneous peaks shown in Figure 22 are also believed to be derived from these target substances/related substances. It is difficult to manufacture a large number of drugs while maintaining the heterogeneity of the target substance/related substance. It is hoped that the antibody molecules developed as drugs are as uniform as possible (with little heterogeneity). Therefore, for wild-type IgG2, when developing antibodies as drugs, there are problems with the important uniformity. In fact, US20060194280 (A1) reported that various miscellaneous peaks from disulfide bonds were observed in ion exchange chromatography analysis of native IgG2, and the biological activities of these peaks were different. As a method for single-stepping the miscellaneous peaks, US20060194280 (A1) reported a method for refolding in the purification step. However, since adopting the above steps in manufacturing increases costs and is complicated, it is preferred to use a method for single-stepping the miscellaneous peaks by amino acid substitution. Although it is believed that when developing IgG2 as a drug, it is hoped to solve the heterogeneity from the disulfide bonds in the hinge region, there is no report on the method for solving this problem by amino acid substitution so far.

Preparation and evaluation of modified CH1 domain and hinge region of WT-IgG2

As shown in Figure 23, it is believed that IgG2 molecules have various disulfide bond conformations. As the cause of the heterogeneity of disulfide bonds from the IgG2 hinge region, it is believed that the inconsistency of disulfide bonds and the presence of free cysteine are the cause. IgG2 has two cysteines (EU numbering positions 219 and 220) in the upper hinge region. As cysteines adjacent to the two cysteines of the upper hinge, there are cysteine at EU numbering position 131 in the CH1 domain of the H chain and cysteine at the C-terminus of the L chain, as well as two cysteines of the same upper hinge of the partner H chain of dimerization. That is, there are a total of 8 cysteines adjacent to each other around the upper hinge of IgG2 in the H2L2 association state, which suggests that there are various heterogeneities due to the inconsistency of disulfide bonds and the presence of free cysteine.

In order to reduce the heterogeneity from the IgG2 hinge region, the hinge region sequence and CH1 domain of IgG2 were modified. Studies were conducted to avoid the heterogeneity caused by the inconsistency of disulfide bonds and free cysteine in IgG2. The results of the various modifications are considered to be: by modifying the cysteine at EU numbering position 131 and the arginine at position 133 present in the H chain CH1 domain in the wild-type IgG2 constant region sequence to serine and lysine respectively and modifying the cysteine at EU numbering position 219 present in the H chain hinge to serine (hereinafter referred to as IgG2-SKSC) (IgG2-SKSC, SEQ ID NO: 120), heterogeneity can be avoided without reducing thermal stability. It is believed that by the above-mentioned modification, the covalent bond between the H chain and the L chain of IgG2-SKSC is uniformly formed by the disulfide bond between the cysteine at EU numbering position 220 and the cysteine at the C-terminus of the L chain (Figure 24).

The expression vector for IgG2-SKSC was prepared, expressed, and purified according to the method described in Example 1. To evaluate the homogeneity of the purified IgG2-SKSC and wild-type IgG2 (WT-IgG2), they were analyzed by cation exchange chromatography.

IEC evaluation method:

System: Waters Alliance

Column: ProPac WCX-10 (Dionex)

Mobile phase A: 25 mM MES-NaOH, pH 5.6

B: 25 mM MES-NaOH, 250 mM sodium acetate, pH 5.6

Flow rate, wavelength: 0.5mL/min, 280nm

Gradient B: 50%-100% (75 min)

The results are shown in Figure 25. As described above, the results show that WT-IgG2 exhibits multiple peaks, while IgG2-SKSC elutes as a single peak. This suggests that the modification introduced into IgG2-SKSC, which forms a single disulfide bond between the cysteine at EU numbering position 220 and the C-terminal cysteine of the L chain, can avoid the heterogeneity of disulfide bonds in the IgG2 hinge region. The thermal denaturation intermediate temperatures of WT-IgG1, WT-IgG2, and IgG2-SKSC were measured using the same method as in Example 5. A peak corresponding to the Fab domain, with a lower Tm value than that of WT-IgG1, was observed in WT-IgG2, whereas no such peak was observed in IgG2-SKSC. This indicates that IgG2-SKSC exhibits superior thermal stability compared to WT-IgG2.

While wild-type IgG2 is known to pose challenges with homogeneity, a crucial aspect in developing antibodies for pharmaceuticals, it has been shown that using IgG2-SKSC as the IgG2 constant region sequence can overcome this issue, making IgG2-SKSC highly useful in developing IgG2 as a pharmaceutical. Furthermore, IgG2-SKSC is also beneficial due to its excellent thermal stability.

[Example 9] Improvement of C-terminal heterogeneity of IgG molecules

Construction of expression vector for WT-IgG1 H chain C-terminal ΔGK antibody

As heterogeneity in the C-terminal sequence of antibodies, there have been reports of C-terminal amino group amidation caused by the loss of a lysine residue at the C-terminal amino acid and the loss of two C-terminal amino acids, glycine and lysine (Johnson KA, Paisley-Flango K, Tangarone BS, Porter TJ, Rouse JC. Cation exchange-HPLC and mass spectrometry reveal C-terminal amidation of an IgG1 heavy chain. Anal Biochem. 2007 Jan 1; 360(1): 75-83). When developing antibodies as pharmaceuticals, it is desirable to eliminate such heterogeneity. In fact, even in the humanized PM-1 antibody TOCILIZUMAB, the main component is a sequence in which the lysine residue at the C-terminal amino acid in the nucleotide sequence is deleted by post-translational modification, and minor components with residual lysine also exist as heterogeneity. Therefore, in order to reduce the heterogeneity of the C-terminal amino acid, the C-terminal amino acid is modified. Specifically, the lysine and glycine at the C-terminus of the H chain constant region of wild-type IgG1 were deleted from the nucleotide sequence to investigate whether the amidation of the C-terminal amino group caused by the deletion of the two C-terminal amino acids, glycine and lysine, could be suppressed.

Using the pB-CH vector of the humanized PM-1 antibody (WT) obtained in Example 1, mutations were introduced into the H chain C-terminal sequence. Using the QuikChange site-directed mutagenesis kit (Stratagene), mutations were introduced into the nucleotide sequence encoding the 447th EU numbering Lys and/or the 446th EU numbering Gly according to the method described in the appendix instructions, turning them into stop codons. This produced expression vectors for an antibody with one C-terminal amino acid, lysine (447th EU numbering), previously deleted, and two C-terminal amino acids, glycine and lysine (446th and 447th EU numbering, respectively), previously deleted. By expressing the L chain of the humanized PM1 antibody, an H chain C-terminal ΔK antibody and an H chain C-terminal ΔGK antibody were obtained. Antibody expression and purification were performed according to the method described in Example 1.

Cation exchange chromatography analysis of the purified H-chain C-terminal ΔGK antibody was performed as follows. The purified H-chain C-terminal ΔGK antibody was analyzed by cation exchange chromatography according to the following method to evaluate the effect of the C-terminal deletion on heterogeneity. The cation exchange chromatography analysis conditions were as follows, and the chromatograms of the humanized PM1 antibody, the H-chain C-terminal ΔGK antibody, and the H-chain C-terminal ΔGK antibody were compared.

Column: ProPac WCX-10, 4×250mm (Dionex)

Mobile phase A: 25 mmol/L MES/NaOH, pH 6.1

B: 25mmol/L MES/NaOH, 250mmol/L NaCl, pH6.1

Flow rate: 0.5 mL/min

Gradient: 25% B (5 min) → (105 min) → 67% B → (1 min) → 100% B (5 min)

Detection: 280nm

The analysis results of the unmodified humanized PM-1 antibody, the H-chain C-terminal ΔK antibody, and the H-chain C-terminal ΔGK antibody are shown in Figure 26. According to the non-patent literature (Chu GC, Chelius D, Xiao G, Khor HK, Coulibaly S, Bondarenko PV. Accumulation of Succinimide in a Recombinant Monoclonal Antibody in Mildly Acidic Buffers Under Elevated Temperatures. Pharm Res. 2007 Mar 24; 24(6): 1145-56), the base peak with a delayed retention time relative to the main peak includes a Lys residue at position 449 and a Pro amide at position 447 at the H-chain C-terminus. While a significant reduction in the base peak was not observed with the H-chain C-terminal ΔK antibody, a significant reduction in the base peak was observed with the H-chain C-terminal ΔGK antibody. This indicates that the H-chain C-terminal heterogeneity can be reduced for the first time by deleting two amino acids at the H-chain C-terminus.

In order to evaluate the effect of the deletion of two residues at the C-terminus of the H chain on thermal stability, the thermal denaturation temperature of the H chain C-terminal ΔGK antibody was measured by DSC. As a DSC measurement, the buffer was replaced by dialysis with 20mM acetate buffer (pH 6.0) containing 150mM NaCl. The humanized PM-1 antibody, the H chain C-terminal ΔGK antibody and the reference solution (dialysis external solution) were fully degassed, and then the above solutions were respectively sealed in a calorimeter box and fully thermally balanced at 40°C. Next, a DSC scan was performed in the range of 40°C to 100°C at a scanning speed of about 1K/min. With reference to non-patent literature (Rodolfo et al., Immunology Letters, 1999, pages 47-52), when the obtained denaturation peak was assigned, it was confirmed that the C-terminal deletion had no effect on the thermal denaturation temperature of the CH3 domain.

Thus, by pre-deleting the C-terminal lysine and glycine from the nucleotide sequence of the H chain constant region, C-terminal amino acid heterogeneity can be reduced without affecting the thermal stability of the antibody. Since the C-terminal sequences of human antibody constant regions IgG1, IgG2, and IgG4 are all Lys at EU numbering position 447 and Gly at EU numbering position 446, the methods for reducing C-terminal amino acid heterogeneity discovered in this example and others are also considered applicable to IgG2 and IgG4 constant regions, or their modified forms.

[Example 10] Preparation of a novel optimized constant region M14ΔGK sequence

Antibody drugs designed to neutralize antigens do not require effector functions such as ADCC in the Fc region, so binding to Fcγ receptors is unnecessary. Binding to Fcγ receptors is not considered preferable from the perspectives of immunogenicity and side effects (Strand V, Kimberly R, Isaacs JD. Biologic therapies in rheumatology: lessons learned, future directions. Nat Rev Drug Discov. 2007 Jan; 6(1): 75-92., Gessner JE, Heiken H, Tamm A, Schmidt RE. The IgG Fc receptor family. Ann Hematol. 1998 Jun; 76(6): 231-48.). Tocilizumab, a humanized anti-IL-6 receptor IgG1 antibody, specifically binds to the IL-6 receptor and neutralizes its biological effects, making it a therapeutic drug for IL-6-related diseases such as rheumatoid arthritis, without requiring binding to Fcγ receptors.

Preparation and evaluation of the Fcγ receptor-unbinding optimized constant region M14ΔGK

As a method to reduce Fcγ receptor binding, one method is to change the isotype of IgG antibodies from IgG1 to IgG2 or IgG4 (Ann. Hematol. 1998 Jun; 76(6): 231-48.) As a method to completely eliminate Fcγ receptor binding, a method has been reported to introduce artificial modifications into the Fc region. For example, since the effector functions of anti-CD3 and anti-CD4 antibodies can cause side effects, amino acid mutations that are not present in the wild-type sequence are introduced into the Fcγ receptor-binding portion of the Fc region (Cole MS, Anasetti C, Tso JY. Human IgG2 variants of chimeric anti-CD3 are nonmitogenic to T cells. J Immunol. 1997 Oct 1; 159(7): 3613-21., Reddy MP, Kinney CA, Chaikin MA, Payne A, Fishman-Lobell J, Tsui P, Dal Monte PR, Doyle ML, Brigham-Burke MR, Anderson D, Reff M, Newman R, Hanna N, Sweet RW, Truneh A. Elimination of Fc receptor-dependent effector functions of a modified IgG4 monoclonal antibody to human CD4. J Immunol. 2000 Feb 20). 15;164(4):1925-33.), clinical trials of the obtained Fcγ receptor-nonbinding anti-CD3 and anti-CD4 antibodies are underway (Strand V, Kimberly R, Isaacs JD. Biologic therapies in rheumatology: lessons learned, future directions. Nat Rev Drug Discov. 2007 Jan;6(1):75-92., Chau LA, Tso JY, Melrose J, Madrenas J. HuM291 (Nuvion), a humanized Fc receptor-nonbinding antibody against CD3, anergizes peripheral blood T cells as partial agonist of the T cell receptor. Transplantation. 2001 Apr 15;71(7):941-50.). It has been reported that by converting the FcγR binding site of IgG1 (EU numbering positions 233, 234, 235, 236, 327, 330, 331) to the sequence of IgG2 (EU numbering positions 233, 234, 235, 236) and IgG4 (EU numbering positions 327, 330, 331), Fcγ receptor-nonbinding antibodies can be produced (Kim SJ, Park Y, Hong HJ., Antibody engineering for the development of therapeutic antibodies., Mol Cells. 2005 Aug 31; 20(1): 17-29. Review.). However, if all of the above mutations are introduced into IgG1, a new 9-amino acid peptide sequence that can form a T cell epitope peptide that does not exist naturally will appear, which increases the risk of immunogenicity. When developing antibodies as pharmaceuticals, it is desirable to have an extremely low risk of immunogenicity.

To address the above issues, modifications to the IgG2 constant region have been investigated. In the FcγR binding site of the IgG2 constant region, EU numbering positions 233, 234, 235, and 236 are non-binding, while EU numbering positions 327, 330, and 331 in the FcγR binding site are different from the non-binding IgG4 sequence. Therefore, it is necessary to convert the amino acids at EU numbering positions 327, 330, and 331 to the IgG4 sequence (G2Δa in Eur. J. Immunol. 1999 Aug; 29(8): 2613-24). However, EU numbering position 339 in IgG4 is alanine, while in IgG2 it is threonine. Therefore, converting only the amino acids at EU numbering positions 327, 330, and 331 to the IgG4 sequence would result in a novel 9-amino acid peptide sequence that can form a T-cell epitope peptide that does not exist naturally, which increases the risk of immunogenicity and is therefore not preferred. Therefore, the present inventors discovered that, in addition to the above modifications, the threonine at EU numbering position 339 of IgG2 was substituted with alanine, thereby preventing the emergence of new peptide sequences.

In addition to the above-mentioned mutations, the following mutations were also introduced: the methionine-to-valine substitution at EU position 397 in IgG2, which was discovered in Example 7 to improve IgG2 stability under acidic conditions; and the cysteine-to-serine substitution at EU position 131, the arginine-to-lysine substitution at EU position 133, and the cysteine-to-serine substitution at EU position 219, which were discovered in Example 8 to improve heterogeneity from the hinge region disulfide bond. Furthermore, since the introduction of mutations at positions 131 and 133 creates a novel 9-amino acid peptide sequence that can form a T-cell epitope peptide not found in nature, posing a risk of immunogenicity, the peptide sequence around positions 131 to 139 was made identical to the naturally occurring human sequence by introducing mutations at positions 131 and 133, which substitute glutamic acid for glycine at EU position 137 and serine for glycine at EU position 138. Furthermore, in order to reduce heterogeneity from the C-terminus, glycine and lysine at positions 446 and 447 of the EU numbering at the C-terminus of the H chain were deleted. The constant region sequence into which all the above mutations were introduced was designated M14ΔGK (M14ΔGK, SEQ ID NO: 24). Although there is a mutation in M14ΔGK in which cysteine at position 219 is substituted with serine, which is a new peptide sequence of 9 amino acids capable of forming a T cell epitope peptide, the risk of immunogenicity is considered to be extremely small due to the similar properties of cysteine and serine as amino acid sequences. No changes in immunogenicity were confirmed in the immunogenicity prediction using TEPITOPE.

An expression vector containing an antibody H chain sequence (M14ΔGK, SEQ ID NO: 24; WT-M14ΔGK, SEQ ID NO: 113) having WT as the variable region sequence and M14ΔGK as the constant region sequence was prepared according to the method described in Example 1. Expression and purification were performed using WT-M14ΔGK as the H chain and WT as the L chain according to the method described in Example 1.

WT-M17ΔGK (M17ΔGK, SEQ ID NO: 116; WT-M17ΔGK, SEQ ID NO: 115) was prepared according to the same method. In the WT-M17ΔGK, mutations were introduced into EU numbering positions 233, 234, 235, 236, 327, 330, 331, and 339 of the IgG1 constant region (G1Δab in Eur. J. Immunol. 1999 Aug; 29(8): 2613-24) to reduce binding to Fcγ receptors, and amino acids at EU numbering positions 446 and 447 were deleted to reduce C-terminal heterogeneity (Example 9). Expression vectors of WT-M11ΔGK (M11ΔGK, SEQ ID NO: 25; WT-M11ΔGK, SEQ ID NO: 114) were prepared. In the WT-M11ΔGK, mutations were introduced into positions 233, 234, 235, and 236 of the IgG4 constant region in order to reduce binding to Fcγ receptors (Eur. J. Immunol. 1999). Aug; 29(8): 2613-24 in G4Δb; in this modification, since a new non-human sequence is generated, the immunogenicity risk is increased); in order to reduce the immunogenicity risk, in addition to the above modifications, mutations were introduced into EU numbering positions 131, 133, 137, 138, 214, 217, 219, 220, 221, and 222 to make the conformation of the hinge region disulfide bond the same as that of M14ΔGK; and a mutation was introduced into EU numbering position 409 to improve stability under acidic conditions (Example 7); amino acids at EU numbering positions 446 and 447 were deleted to reduce C-terminal heterogeneity (Example 9). WT-M17ΔGK or WT-M11ΔGK was used as the H chain and WT was used as the L chain, and expression and purification were performed according to the method described in Example 1.

Evaluation of Fcγ receptor binding activity of WT-M14ΔGK, WT-M17ΔGK, and WT-M11ΔGK

Binding to FcγRI was evaluated as follows. Using Biacore T100, human Fcγ receptor I (hereinafter referred to as FcγRI) immobilized on a sensor chip was allowed to interact with IgG1, IgG2, IgG4, M11ΔGK, M14ΔGK, and M1ΔGK 7 used as analytes, and the binding amounts were compared. Recombinant human FcRIA/CD64 (R&D Systems) was used for human FcγRI, and IgG1, IgG2, IgG4, M11ΔGK, M14ΔGK, and M17ΔGK were used as samples for measurement. FcγRI was immobilized on a sensor chip CM5 (BIACORE) using an amine coupling method. The final immobilization amount of hFcγRI was approximately 13,000 RU (resonance units). HBS-EP+ was used as the running buffer, and the flow rate was 20 μL/min. The sample was adjusted to a concentration of 100 μg/mL using HBS-EP+. The analysis consists of two steps: a 2-minute association phase, during which 10 μL of the antibody solution is injected, followed by a 4-minute dissociation phase, during which the injection solution is switched to HBS-EP+. After the dissociation phase, 20 μL of 5 mM sodium hydroxide is injected to regenerate the sensor chip. This cycle of binding, dissociation, and regeneration constitutes one analysis cycle, during which various antibody solutions are injected to generate sensorgrams. IgG4, IgG2, IgG1, M11, M14, and M17 were injected as analytes, and this process was repeated twice. A comparison of the measured binding data is shown in Figure 27. The results show that the binding amount decreases in the order of IgG1 > IgG4 > IgG2 = M11ΔGK = M14ΔGK = M17ΔGK, demonstrating that wild-type IgG2, M11ΔGK, M14ΔGK, and M17ΔGK bind weaker to FcγRI than wild-type IgG1 and IgG4.

The evaluation of binding to FcγRIIa was performed as follows. Using Biacore T100, the human-derived Fcγ receptor IIa (hereinafter referred to as FcγRIIa) fixed on the sensor chip was interacted with IgG1, IgG2, IgG4, M11ΔGK, M14ΔGK, and M17ΔGK used as analytes, and then the binding amounts were compared. Human-derived FcγRIIa was measured using recombinant human FcRIIA/CD32a (R&D Systems) and IgG1, IgG2, IgG4, M11ΔGK, M14ΔGK, and M17ΔGK as samples. FcγRIIa was fixed on the sensor chip CM5 (BIACORE) using an amine coupling method. The final fixed amount of FcγRIIa was approximately 3300RU. The running buffer used was HBS-EP+ at a flow rate of 20 μL/min. Afterwards, the running buffer was injected until the baseline stabilized, and the measurement was started after the baseline stabilized. Each IgG isotype (IgG1, IgG2, IgG4) and an antibody with a mutation introduced (M11ΔGK, M14ΔGK, M17ΔGK) as the analyte was allowed to interact with the immobilized FcγRIIa and observe its binding amount. HBS-EP+ was used as the running buffer, the flow rate was 20 μL/min, and the measurement temperature was 25°C. Each IgG and modified body was adjusted to 100 μg/mL, and 20 μL of analyte was injected to interact with the immobilized FcγRIIa. After the interaction, 200 μL of running buffer was injected to dissociate the analyte from FcγRIIa and regenerate the sensor chip. Analytes IgG4, IgG2, IgG1, M11ΔGK, M14ΔGK, and M17ΔGK were injected in sequence and the operation was repeated twice. The comparison results of the measured binding amount data are shown in Figure 28. The results showed that the binding amount decreased in the order of IgG1 > IgG2 = IgG4 > M11ΔGK = M14ΔGK = M17ΔGK, indicating that M11ΔGK, M14ΔGK, and M17ΔGK bind weakly to FcγRIIa compared to wild-type IgG1, IgG2, and IgG4.

The evaluation of binding to FcγRIIb was performed as follows. Using Biacore T100, the Fcγ receptor IIb (hereinafter referred to as FcγRIIb) derived from humans and fixed on the sensor chip was interacted with IgG1, IgG2, IgG4, M11ΔGK, M14ΔGK, and M17ΔGK used as analytes, and the binding amounts were compared. Recombinant human FcRIIB/C (R&D Systems) was used for human FcγRIIb, and IgG1, IgG2, IgG4, M11ΔGK, M14ΔGK, and M17ΔGK were used as samples for measurement. FcγRIIb was fixed to the sensor chip CM5 (BIACORE) by the amine coupling method, and the final fixed amount of FcγRIIb was about 4300RU. Afterwards, the running buffer was injected until the baseline was stable, and the measurement was started after the baseline was stable. Analytes of various IgG isotypes (IgG1, IgG2, and IgG4) and mutated antibodies (M11ΔGK, M14ΔGK, and M17ΔGK) were allowed to interact with immobilized FcγRIIb, and the binding capacity was measured. The running buffer used was HBS-EP+ (10 mM HEPES, 0.15 M NaCl, 3 mM EDTA, and 0.05% v/v Surfactant P20), with a flow rate of 20 μL/min and a measurement temperature of 25°C. Each IgG and its modified form was adjusted to 200 μg/mL, and 20 μL of the analyte was injected to allow interaction with the immobilized FcγRIIb. After interaction, 200 μL of running buffer was injected to dissociate the analyte from FcγRIIb and regenerate the sensor chip. The analytes IgG4, IgG2, IgG1, M11ΔGK, M14ΔGK, and M17ΔGK were injected in this order, and this procedure was repeated twice. Comparison of the measured binding data is shown in Figure 29. The results show that the binding amount decreases in the order of IgG4 > IgG1 > IgG2 > M11ΔGK = M14ΔGK = M17ΔGK, indicating that M11ΔGK, M14ΔGK, and M17ΔGK bind weaker to FcγRIIb than wild-type IgG1, IgG2, and IgG4.

Binding to FcγRIIIa was evaluated as follows. Using Biacore T100, human Fcγ receptor IIIa (hereinafter referred to as FcγRIIIa) immobilized on a sensor chip was allowed to interact with analytes IgG1, IgG2, IgG4, M11ΔGK, M14ΔGK, and M17ΔGK, and the binding amounts were compared. hFcγRIIIaV-His6 (recombinant hFcγRIIIaV-His6: in-house product) was used for human FcγRIIIa, and IgG1, IgG2, IgG4, M11ΔGK, M14ΔGK, and M17ΔGK were used as samples for the assay. FcγRIIIa was immobilized on a sensor chip CM5 (BIACORE) using an amine coupling method, with the final immobilized amount of hFcγRIIIaV-His6 being approximately 8200 RU (resonance units). HBS-EP+ was used as the running buffer, and the flow rate was 5 μL/min. The sample concentration was adjusted to 250 μg/mL using HBS-EP+. The analysis consisted of two steps: a 2-minute association phase, during which 10 μL of the antibody solution was injected. This was followed by a 4-minute dissociation phase, during which the injection solution was switched to HBS-EP+. After the dissociation phase, the sensor chip was regenerated by injecting 20 μL of 5 mM hydrochloric acid. This binding, dissociation, and regeneration cycle constituted one analysis cycle. Sensorgrams were generated by injecting various antibody solutions. Analytes were injected in this order: IgG4, IgG2, IgG1, M11ΔGK, M14ΔGK, and M17ΔGK. A comparison of the measured binding data is shown in Figure 30. The results show a decreasing order of IgG1>>IgG4>IgG2>M17ΔGK>M11ΔGK=M14ΔGK, indicating that M11ΔGK, M14ΔGK, and M17ΔGK bind weakly to FcγRIIIa compared to wild-type IgG1, IgG2, and IgG4. Furthermore, it was found that M11ΔGK and M14ΔGK bind to FcγRIIIa more weakly than M17ΔGK containing the G1Δab mutation reported in Eur. J. Immunol. 1999 Aug; 29(8): 2613-24.

These results confirm that WT-M14ΔGK, WT-M17ΔGK, and WT-M11ΔGK exhibit significantly reduced binding to various Fcγ receptors compared to wild-type IgG1. Using WT-M14ΔGK, WT-M17ΔGK, and WT-M11ΔGK as constant regions can avoid the risk of immunogenicity due to Fcγ receptor-mediated internalization into APCs and side effects caused by effector functions such as ADCC. Therefore, WT-M14ΔGK, WT-M17ΔGK, and WT-M11ΔGK are useful constant region sequences for antibody therapeutics intended for antigen neutralization.

High-concentration stability test of WT-M14ΔGK, WT-M17ΔGK, and WT-M11ΔGK

The stability of WT-M14ΔGK, WT-M17ΔGK, and WT-M11ΔGK in high-concentration formulations was evaluated. Purified antibodies of WT-IgG1, WT-M14ΔGK, WT-M17ΔGK, and WT-M11ΔGK were dialyzed against 20 mM histidyl chloride, 150 mM NaCl, pH 6.5 (EasySEP, TOMY), then concentrated using an ultrafiltration membrane for high-concentration stability testing. The experimental conditions are as follows.

Antibodies: WT-IgG1, WT-M14ΔGK, WT-M17ΔGK, WT-M11ΔGK

Buffer: 20 mM histidyl chloride, 150 mM NaCl, pH 6.5

Concentration: 61 mg/mL

Storage temperature and storage time: 40°C for 2 weeks, 40°C for 1 month, 40°C for 2 months

Aggregate evaluation method:

System: Waters Alliance

Column: G3000SWxl (TOSOH)

Mobile phase: 50 mM sodium phosphate, 300 mM KCl, pH 7.0

Flow rate, wavelength: 0.5mL/min, 220nm

The samples were diluted 100-fold before analysis

The aggregate content of the original preparation (freshly prepared preparation) and preparations stored under various conditions was evaluated using the gel filtration chromatography method described above. The change in aggregate content in the original preparation is shown in Figure 31. The results showed that the increase in aggregate content in WT-M14ΔGK, WT-M17ΔGK, and WT-M11ΔGK was low compared to WT-IgG1, approximately half the increase in aggregate content in WT. As shown in Figure 32, the increase in Fab fragment content was similar between WT-IgG1 and WT-M17ΔGK, but the increase in Fab fragment content in WT-M14ΔGK and WT-M11ΔGK was approximately one-quarter of the increase in Fab fragment content in WT. As described in WO 2003/039485, the degeneration pathway of IgG antibody preparations primarily involves the formation of aggregates and Fab degradation products. The study found that WT-M14ΔGK and WT-M11ΔGK exhibited superior formulation stability compared to WT-IgG1 in terms of both aggregate formation and Fab fragment production. This suggests that even for antibodies whose IgG1 constant regions lack sufficient stability, preventing them from being formulated into high-concentration solutions suitable for pharmaceutical development, using WT-M14ΔGK, WT-M17ΔGK, or WT-M11ΔGK as constant regions could enable the production of highly stable, high-concentration solution formulations.

In particular, M14ΔGK is considered extremely useful as a novel constant region sequence that improves the instability of native IgG2 molecules under acidic conditions, improves heterogeneity derived from hinge region disulfide bonds, does not bind to Fcγ receptors, minimizes the generation of a novel 9-amino acid peptide sequence capable of forming a T cell epitope peptide, and exhibits superior stability compared to IgG1 in high-concentration preparations.

[Example 11] Preparation of PF1-M14ΔGK Antibody

The variable region of PF1 (IgG1 constant region) produced in Example 5 was excised using XhoI/NheI, and the constant region of M14ΔGK (WT variable region) produced in Example 7 was excised using NheI/NotI. These two H chain antibody gene fragments were inserted into an animal cell expression vector to create the desired PF1-M14ΔGK H chain expression vector (PF1_H-M14ΔGK, SEQ ID NO: 117). PF1_L was used as the L chain, and the PF1-M14ΔGK antibody was expressed and purified according to the methods described in Example 1.

The PF1-M14ΔGK antibody is superior to the WT (humanized PM-1 antibody) in all aspects as an anti-IL-6 receptor antibody drug and is considered to be extremely useful.

[Example 12] Preparation and evaluation of M31ΔGK

M31ΔGK (M31ΔGK, SEQ ID NO: 118) was prepared by converting the amino acids 330, 331, and 339 of the EU numbering sequence of M14ΔGK prepared in Example 10 to those of an IgG2 sequence. An expression vector containing the antibody H chain sequence (WT-M31ΔGK, SEQ ID NO: 119) containing WT as the variable region sequence and M31ΔGK as the constant region sequence was prepared according to the method described in Example 1. WT-M31ΔGK was used as the H chain and WT as the L chain, and WT-M31 was expressed and purified according to the method described in Example 1.

In addition to WT-M31, WT-IgG2 and WT-M14ΔGK, which were co-expressed and purified, were analyzed by cation exchange chromatography as follows. The cation exchange chromatography analysis conditions were as follows, and the chromatograms of WT-IgG2, WT-M14ΔGK, and WT-M31ΔGK were compared.

Column: ProPac WCX-10, 4×250mm (Dionex)

Mobile phase A: 25 mmol/L MES/NaOH, pH 6.1

B: 25mmol/L MES/NaOH, 250mmol/L NaCl, pH6.1

Flow rate: 0.5 mL/min

Gradient: 0% B (5 min) → (65 min) → 100% B → (1 min)

Detection: 280nm

The analysis results for WT-IgG2, WT-M14ΔGK, and WT-M31ΔGK are shown in Figure 33. As shown, WT-IgG2 exhibits multiple peaks, while WT-M31ΔGK and WT-M14ΔGK elute as a single peak. This suggests that the heterogeneity of the hinge disulfide bonds in IgG2 is also avoided in WT-M31ΔGK.

[Example 13] Preparation of fully humanized antibody F2H/L39-IgG1

Full humanization of the framework sequence of the PF1 antibody

In PF1_H prepared in Example 5, only the arginine at position 71 (Kabat numbering; Kabat EA et al. 1991. Sequence of Proteins of Immunological Interest. NIH) remains intact in the mouse sequence, making this undesirable from an immunogenicity perspective. Residue 71 of the H chain is generally a key sequence determining the HCDR2 structure. In fact, it has been reported that, when humanizing PM1 antibodies were produced, residue 71 is crucial for the binding activity of mouse PM1 antibodies. Substitution of arginine at position 71 with valine has been shown to significantly decrease binding activity (Cancer Research 53, 851-856, 1993). Similarly, PF1_H is classified as a member of the VH4 family of human germline genes, and valine is highly conserved at position 71 within the VH4 family. Substitution of arginine at position 71 with valine has been shown to significantly decrease neutralizing activity.

Therefore, in order to retain the arginine residue at position 71 while completely removing the mouse sequence, the present inventors studied human germline genes and reported human antibody sequences, searching for a sequence that contains arginine at position 71 and retains residues important for maintaining the antibody's three-dimensional structure. As a result, a candidate sequence was identified that, while showing low homology to PF1_H shown in Table 9, retained the important residues.

[Table 9]

H96-IgG1 (SEQ ID NO: 134 amino acid sequence) was designed by substituting the above candidate sequence from positions 66 to 94 of the Kabat numbering of PF1_H-IgG1. The variable region of the antibody was generated by PCR combined with synthetic oligoDNA (assembly PCR). The constant region was amplified by PCR from an IgG1 expression vector. Assembly PCR was used to bind the variable and constant regions and then insert them into an animal cell expression vector. Expression and purification of H96/PF1L-IgG1 were performed according to the methods described in Example 1.

Evaluation of the fully humanized antibody H96/PF1L-IgG1

Using purified H96/PF1L-IgG1, Tm values were determined according to the method described in Example 5. Affinity measurements were performed under the same conditions as in Example 5, except that the SR344 concentration was adjusted to 0, 0.36, and 1.4 μg/mL, and the dissociation phase was measured for 15 minutes. The results showed that H96/PF1L-IgG1 exhibited Tm values and affinity comparable to those of PF1-IgG1 (Table 10).

[Table 10]

Tm(℃) KD(M) PF1 antibody 91.3 1.4E+06 4.2E-05 3.1E-11 H96/PF1L-IgG1 89.8 1.2E+06 4.8E-05 3.9E-11

As described above, by using H96 as the H chain of the PF1 antibody, the remaining mouse sequences in the PF1 antibody were completely removed while maintaining the Tm value and affinity, resulting in a fully humanized antibody framework. Since the framework sequence of H96/PF1L-IgG1 contains no mouse-derived sequences, H96/PF1L-IgG1 is believed to be particularly superior from the perspective of immunogenicity.

Production of F2H/L39-IgG1 with reduced pI and immunogenicity risk

As shown in Example 4, it has been demonstrated that plasma retention can be improved by modifying the amino acids in the antibody variable region and lowering the pI. Therefore, the following amino acid substitutions were further introduced into the H96-IgG1 prepared above. To lower the pI, lysine at position 64 was substituted with glutamine, and glycine at position 65 was substituted with aspartic acid. Furthermore, to reduce the risk of immunogenicity, glutamic acid at position 105 was substituted with glutamine, and threonine at position 107 was substituted with isoleucine. Furthermore, as affinity-enhancing modifications obtained in Example 2, valine at position 95 was substituted with leucine, and isoleucine at position 99 was substituted with alanine. Following the method described in Example 1, the above amino acid substitutions were introduced into H96-IgG1 to produce F2H-IgG1 (SEQ ID NO: 135 (amino acid sequence)).

The following amino acid substitutions were introduced into PF1L. To lower the pI, glutamine at position 27 was substituted with glutamic acid, and leucine at position 55 was substituted with glutamic acid. These amino acid substitutions were introduced into PF1L according to the method described in Example 1 to produce L39 (SEQ ID NO: 136 (amino acid sequence)). F2H/L39-IgG1 was expressed and purified using the method described in Example 1.

Biacore analysis of the affinity of F2H/L39-IgG1 for human IL-6 receptor

The affinities of humanized PM1 antibody (wild-type, WT), PF1 antibody (produced in Example 5), and F2H/L39-IgG1 were measured. These measurements were performed under the same conditions as in Example 4. However, the concentrations of SR344 were adjusted to 0, 0.36, and 1.4 μg/mL, and the dissociation phase was measured for 15 minutes (Table 11).

[Table 11]

The results showed that the KD value of F2H/L39-IgG1 remained at the order of ^10-11 , indicating extremely strong affinity. However, compared with PF1-IgG1, its _ka value dropped to about 1/2.

Evaluation of the human IL-6 receptor neutralizing activity of F2H/L39-IgG1

The neutralizing activity of humanized PM1 antibody (wild-type, WT) and F2H/L39-IgG1 was evaluated according to the method described in Example 1. Neutralization activity was assessed at a human interleukin-6 (TORAY) concentration of 600 ng/mL. As shown in Figure 34 , F2H/L39-IgG1 exhibited significantly stronger activity, over 100-fold higher than that of WT at a 100% inhibitory concentration.

Evaluation of the isoelectric point of F2H/L39-IgG1 by isoelectric electrophoresis

The isoelectric point of F2H/L39-IgG1 was measured according to the method described in Example 3. The isoelectric point of F2H/L39-IgG1 was 5.5, which was significantly lower than that of the PF1 antibody prepared in Example 5, resulting in further improved plasma retention.

The theoretical isoelectric point of the F2H/L39 variable region (VH and VL sequences) was calculated using GENETYX (GENETYX CORPORATION) to be 4.3. The theoretical isoelectric point of the WT is 9.20. Therefore, by making amino acid substitutions in the WT, the theoretical isoelectric point of the variable region of F2H/L39 was lowered by approximately 4.9.

PK/PD study of F2H/L39-IgG1 in cynomolgus monkeys

The pharmacokinetics (PK) and pharmacodynamics (PD) of humanized PM1 antibody (wild type, WT), PF1 antibody and F2H/L39-IgG1 in cynomolgus monkeys were evaluated. WT, PF1 and F2H/L39-IgG1 were administered subcutaneously at 1.0 mg/kg, and blood was collected over time before and after administration. The plasma concentration of each antibody was determined in the same manner as in Example 6. The changes in the plasma concentrations of WT, PF1 and F2H/L39-IgG1 are shown in Figure 35. In order to evaluate the efficacy of each antibody in neutralizing the membrane-type cynomolgus monkey IL-6 receptor, cynomolgus monkey IL-6 was administered subcutaneously to the lower back at 5 μg/kg on consecutive days from the 3rd to the 10th day after antibody administration, and the CRP concentration of each individual was measured 24 hours later. The changes in CRP concentration after WT and F2H/L39 administration are shown in Figure 36. To evaluate the efficacy of each antibody in neutralizing soluble cynomolgus monkey IL-6 receptor, the concentration of unbound soluble cynomolgus monkey IL-6 receptor in cynomolgus monkey plasma was measured. The changes in the concentration of unbound soluble cynomolgus monkey IL-6 receptor after administration of WT and F2H/L39 are shown in Figure 37.

These results indicate that the plasma concentrations of WT and PF1 varied substantially, and that F2H/L39-IgG1, with its lower pI compared to both antibodies, maintained high plasma concentrations. Furthermore, F2H/L39-IgG1, which has a strong affinity for the IL-6 receptor, maintained lower CRP and unbound soluble cynomolgus monkey IL-6 receptor concentrations compared to WT.

[Example 14] Evaluation of plasma retention of WT-M14

Method for predicting plasma retention in humans

IgG molecules have a long retention time in plasma (and a slow elimination rate) due to the role of FcRn, known as a salvage receptor for IgG molecules (Nat. Rev. Immunol. 2007 Sep;7(9):715-25). IgG molecules that enter endosomes via pinocytosis bind to FcRn expressed within the endosomes under the acidic conditions (around pH 6.0). IgG molecules that fail to bind to FcRn enter lysosomes and are degraded by them. However, IgG molecules that bind to FcRn migrate to the cell surface and dissociate from FcRn under the neutral conditions (around pH 7.4) of plasma, returning to the plasma.

IgG antibodies are known to have IgG1, IgG2, IgG3, and IgG4 isotypes. It has been reported that the half-lives of these isotypes in human plasma are approximately 36 days for IgG1 and IgG2, approximately 29 days for IgG3, and 16 days for IgG4 (Nat. Biotechnol. 2007 Dec; 25(12): 1369-72). IgG1 and IgG2 are believed to have the longest plasma retention. Antibody drugs are generally of the IgG1, IgG2, and IgG4 isotypes. As a method for further improving the plasma retention of these IgG antibodies, a method has been reported to improve the binding activity to human FcRn by modifying the sequence of the IgG constant region (J. Biol. Chem. 2007 Jan 19; 282(3): 1709-17; J. Immunol. 2006 Jan 1; 176(1): 346-56).

Since there are species-specific differences between mouse FcRn and human FcRn (Proc. Natl. Acad. Sci. USA. 2006 Dec 5; 103(49): 18709-14), in order to predict the plasma retention of IgG antibodies with modified constant region sequences in humans, it is considered necessary to evaluate binding to human FcRn and plasma retention in human FcRn transgenic mice (Int. Immunol. 2006 Dec; 18(12): 1759-69).

Evaluation of binding to human FcRn

FcRn is a complex of FcRn and β2-microglobulin. OligoDNA primers were prepared based on the published human FcRn gene sequence (J. Exp. Med. (1994) 180 (6), 2377-2381). Using human cDNA (Human Placenta Marathon-Ready cDNA, Clontech) as a template, the prepared primers were used to adjust the DNA fragment encoding the full-length gene by PCR. Using the obtained DNA fragment as a template, a DNA fragment encoding the extracellular region (Met1-Leu290) including the signal region was amplified by PCR and then inserted into an animal cell expression vector (human FcRn amino acid sequence, SEQ ID NO: 140). Similarly, oligoDNA primers were prepared based on the published human β2-microglobulin gene sequence (Proc. Natl. Acad. Sci. USA. 99 (26), 16899-16903 (2002)). A DNA fragment encoding the full-length gene was prepared by PCR using human cDNA (Hu-Placenta Marathon-Ready cDNA, CLONTECH) as a template and the prepared primers. A DNA fragment encoding the full-length β2-microglobulin (Met1-Met119), including the signal region, was amplified by PCR using this DNA fragment as a template. This fragment was then inserted into an animal cell expression vector (human β2-microglobulin amino acid sequence, SEQ ID NO: 141).

Soluble human FcRn expression was performed according to the following procedure. Human FcRn and human β2-microglobulin plasmids were transfected into HEK293H cells (Invitrogen) derived from human embryonic renal cell carcinoma cells using lipofection with 10% fetal bovine serum (Invitrogen). The resulting culture supernatant was recovered and purified using IgG Sepharose 6 Fast Flow (Amersham Biosciences) according to the method described in (J. Immunol. 2002 Nov 1; 169(9): 5171-80). Purification was then performed using HiTrap Q HP (GE Healthcare).

To evaluate binding to human FcRn, a Biacore 3000 was used. Human FcRn, the analyte, interacted with an antibody bound to protein L or rabbit anti-human IgGκ chain antibody immobilized on a sensor chip. The affinity (KD) was calculated from the amount of human FcRn bound during the interaction. Specifically, protein L or rabbit anti-human IgGκ chain antibody was immobilized on a sensor chip CM5 (BIACORE) using an amine coupling method using 50mM sodium phosphate buffer (pH 6.0) containing 150mM NaCl as the running buffer. The antibody was then diluted with a running buffer containing 0.02% Tween 20 and injected to allow binding to the chip. Human FcRn was then injected to evaluate the binding activity of the antibody to human FcRn [0].

Affinity calculations were performed using BIA evaluation software. The amount of hFcRn bound to the antibody immediately before the end of human FcRn infusion was determined from the resulting sensorgrams. The affinity of the antibody for human FcRn was then calculated by fitting using the steady-state affinity method.

Evaluation of plasma retention in human FcRn transgenic mice

The in vivo kinetics of human FcRn transgenic mice (B6.mFcRn-/-.hFcRn Tg line 276+/+ mice; Jackson Laboratories) were evaluated as follows. The antibody was administered intravenously to mice at a dose of 1 mg/kg, and blood was collected at appropriate time points. The collected blood was immediately centrifuged at 15,000 rpm for 15 minutes at 4°C to obtain plasma. The separated plasma was kept in a refrigerator set to below -20°C until the measurement was performed. The concentration in plasma was determined by ELISA.

Predictive evaluation of WT-M14 plasma retention in humans

The binding activities of WT-IgG1 and WT-M14 to human FcRn were evaluated using BIAcore. The results are shown in Table 12. Compared with WT-IgG1, the binding activity of WT-M14 was slightly stronger.

[Table 12]

However, the plasma retention of WT-IgG1 and WT-M14 in human FcRn transgenic mice was evaluated. As shown in Figure 38, WT-IgG1 and WT-M14 showed equivalent plasma retention. This suggests that the M14 constant region also shows plasma retention equivalent to that of the IgG1 constant region in humans.

[Example 15] Preparation of WT-M44, WT-M58, and WT-M73 with Improved Plasma Retention

Preparation of WT-M58 molecules

As shown in Example 14, WT-M14 exhibited plasma retention comparable to that of WT-IgG1 in human FcRn transgenic mice. While methods for improving plasma retention include lowering the antibody isoelectric point and enhancing FcRn-binding activity, the following modifications were introduced to enhance the plasma retention of WT-M14. Specifically, valine at EU numbering position 397 of WT-M31ΔGK, prepared from WT-M14 in Example 4, was substituted with methionine, histidine at position 268 with glutamine, arginine at position 355 with glutamine, and glutamine at position 419 with glutamic acid. These four modifications were introduced into WT-M31ΔGK to produce WT-M58 (SEQ ID NO: 142 (amino acid sequence)). The expression vector was prepared according to the method described in Example 1. WT-M58 was used as the H chain, and L (WT) was used as the L chain. WT-M58 was expressed and purified according to the methods described in Example 1.

Preparation of WT-M73 molecules

On the other hand, the amino acid at position 434 of EU numbering of IgG1 was substituted with alanine to produce WT-M44 (SEQ ID NO: 143 (amino acid sequence)). The glycine at position 446 and the lysine at position 447 of M44 were deleted to reduce the heterogeneity of the H chain C-terminus to produce WT-M83 (SEQ ID NO: 185 (amino acid sequence)). The amino acid at position 434 of EU numbering of WT-M58 was substituted with alanine to produce WT-M73 (SEQ ID NO: 144 (amino acid sequence)).

The expression vectors for the above antibodies were prepared according to the method of Example 1, using WT-M44, WT-M58, or WT-M73 as the H chain and L (WT) as the L chain. WT-M44, WT-M58, and WT-M73 were expressed and purified according to the method described in Example 1.

Predictive evaluation of plasma retention of WT-M44, WT-M58, and WT-M73 in humans

The binding activity of WT-IgG1, WT-M44, WT-M58, and WT-M73 to human FcRn was evaluated using BIAcore. The results are shown in Table 13. The binding activities of WT-M44, WT-M58, and WT-M73 were all superior to that of WT-IgG1, which were approximately 2.7 times, approximately 1.4 times, and approximately 3.8 times that of WT-IgG1, respectively.

[Table 13]

The plasma retention of WT-IgG1, WT-M44, and WT-M58 in human FcRn transgenic mice was evaluated. As shown in Figure 39 , it was confirmed that WT-M58 had improved plasma retention compared to WT-IgG1 and WT-M44. Furthermore, the plasma retention of WT-IgG1, WT-M44, WT-M58, and WT-M73 in human FcRn transgenic mice was evaluated. As shown in Figure 40 , it was confirmed that the plasma retention of WT-M44, WT-M58, and WT-M73 was improved compared to WT-IgG1, and the improvement in plasma retention correlated with human FcRn binding ability. The plasma concentration of WT-M73 after 28 days was approximately 16-fold higher than that of WT-IgG1. This suggests that, even in humans, antibodies harboring the M73 constant region have significantly improved plasma retention compared to antibodies harboring the IgG1 constant region.

[Example 16] Effects of the novel constant regions M14 and M58 on reducing heterogeneity in various antibodies

As shown in Example 8, in the humanized PM1 antibody (WT), an anti-IL-6 receptor antibody, it was confirmed that the heterogeneity of the hinge region from IgG2 could be reduced by changing the constant region from IgG2 to M14. Therefore, in addition to the humanized PM1 antibody, studies were conducted to investigate whether heterogeneity could be reduced by changing the constant region to M14 or M58 in IgG2 antibodies.

As antibodies other than the humanized PM1 antibody, the anti-IL-6 receptor antibody F2H/L39 (F2H/L39_VH amino acid sequence, SEQ ID NO: 145; F2H/L39_VL amino acid sequence, SEQ ID NO: 146), the anti-IL-31 receptor antibody H0L0 (H0L0_VH amino acid sequence, SEQ ID NO: 147; H0L0_VL amino acid sequence, SEQ ID NO: 148), and the anti-RANKL antibody DNS (DNS_VH amino acid sequence, SEQ ID NO: 149; DNS_VL amino acid sequence, SEQ ID NO: 150) were used. For each antibody, antibodies were prepared whose constant regions were IgG1 (SEQ ID NO: 19), IgG2 (SEQ ID NO: 20), and M14 (SEQ ID NO: 24) or M58 (SEQ ID NO: 151).

As a method for evaluating heterogeneity, cation exchange chromatography was used for evaluation. When evaluating the heterogeneity of the prepared antibodies, a ProPac WCX-10 column (Dionex) was used, 20 mM sodium acetate (pH 5.0) was used as mobile phase A, and 20 mM sodium acetate/1 M NaCl (pH 5.0) was used as mobile phase B. Evaluation was performed using an appropriate flow rate and gradient. The results of the evaluation by cation exchange chromatography (IEC) are shown in Figure 41.

As shown in Figure 41, it was confirmed that heterogeneity increased in the humanized PM1 antibody (WT), an anti-IL-6 receptor antibody, as well as in F2H/L39, an anti-IL-6 receptor antibody, H0L0, an anti-IL-31 receptor antibody, and DNS, an anti-RANKL antibody, by converting the constant region from IgG1 to IgG2. Heterogeneity was reduced in all antibodies by converting the constant region to M14 or M58. This suggests that substitution of cysteine at EU numbering position 131 in the CH1 domain of the H chain and cysteine at EU numbering position 219 in the hinge of the H chain with serine can reduce heterogeneity from native IgG2, regardless of the antibody sequence of the variable region and the type of antigen.

[Example 17] Effect of the Novel Constant Region M58 on Improving Plasma Retention of Various Antibodies

As shown in Example 15, in the humanized PM1 antibody (WT), an anti-IL-6 receptor antibody, it was found that by changing the constant region from IgG1 to M58, the binding activity to human FcRn was enhanced, and the plasma retention in human FcRn transgenic mice was improved. Therefore, in addition to the humanized PM1 antibody, studies were conducted to investigate whether plasma retention could be improved by changing the constant region to M58 for IgG1 antibodies.

As antibodies other than the humanized PM1 antibody (WT), H0L0 (H0L0_VH amino acid sequence, SEQ ID NO: 147; H0L0_VL amino acid sequence, SEQ ID NO: 148), an anti-IL-31 receptor antibody, and DNS (DNS_VH amino acid sequence, SEQ ID NO: 149; DNS_VL amino acid sequence, SEQ ID NO: 150), an anti-RANKL antibody, were used. For each antibody, antibodies with IgG1 (SEQ ID NO: 19) and M58 (SEQ ID NO: 151) constant regions were prepared, and their binding activity to human FcRn was evaluated according to the method described in Example 14. The results are shown in Table 14.

[Table 14]

As shown in Table 14, even in H0L0, an anti-IL-31 receptor antibody, and DNS, an anti-RANKL antibody, human FcRn binding activity was confirmed to be enhanced by converting the constant region from IgG1 to M58, similar to the anti-IL-6 receptor antibody WT. This suggests that, regardless of the antibody variable region sequence and antigen type, converting the constant region from IgG1 to M58 can improve plasma retention in humans.

[Example 18] Effect of cysteine in the CH1 domain on heterogeneity and stability

As shown in Example 8, to reduce the heterogeneity of native IgG2, cysteine residues in the IgG2 hinge and in the CH1 domain were modified. After studying various modifications, it was discovered that heterogeneity could be reduced without compromising stability by using SKSC (SEQ ID NO: 154), a constant region obtained by substituting cysteine at EU numbering position 131 and arginine at EU numbering position 133 in the CH1 domain of the H chain with serine and lysine, respectively, and substituting cysteine at EU numbering position 219 in the hinge of the H chain with serine, in the wild-type IgG2 constant region sequence.

On the other hand, as a method for reducing heterogeneity, there are methods of substituting only the cysteine at position 219 present in the hinge of the H chain with serine, and methods of substituting only the cysteine at position 220 with serine. A constant region SC (SEQ ID NO: 155) in which the cysteine at position 219 of the EU numbering of IgG2 was substituted with serine, and a constant region CS (SEQ ID NO: 156) in which the cysteine at position 220 of the EU numbering of IgG2 was substituted with serine were prepared. Subsequently, WT-SC (SEQ ID NO: 157) and WT-CS (SEQ ID NO: 158) having SC and CS as constant regions, respectively, were prepared, and heterogeneity and thermal stability were compared with WT-IgG1, WT-IgG2, WT-SKSC, and WT-M58. In addition, F2H/L39-IgG1, F2H/L39-IgG2, F2H/L39-SC, F2H/L39-CS, F2H/L39-SKSC, and F2H/L39-M14, which have constant regions IgG1 (SEQ ID NO: 19), IgG2 (SEQ ID NO: 20), SC (SEQ ID NO: 155), CS (SEQ ID NO: 156), SKSC (SEQ ID NO: 154), and M14 (SEQ ID NO: 24), respectively, were prepared from an anti-IL-6 receptor antibody different from WT, namely F2H/L39 (F2H/L39_VH amino acid sequence, SEQ ID NO: 145; F2H/L39_VL amino acid sequence, SEQ ID NO: 146), and the heterogeneity and stability of these antibodies were compared.

Heterogeneity of WT-IgG1, WT-IgG2, WT-SC, WT-CS, WT-SKSC, WT-M58, F2H/L39-IgG1, F2H/L39-IgG2, F2H/L39-SC, F2H/L39-CS, F2H/L39-SKSC, and F2H/L39-M14 was evaluated by cation exchange chromatography. A ProPac WCX-10 (Dionex) column was used, with 20 mM sodium acetate (pH 5.0) as mobile phase A and 20 mM sodium acetate/1 M NaCl (pH 5.0) as mobile phase B. Evaluation was performed using appropriate flow rates and gradients. The results of the cation exchange chromatography evaluation are shown in Figure 42.

As shown in Figure 42, in both WT and F2H/L39, heterogeneity increased when the constant region was changed from IgG1 to IgG2, but significantly decreased when the constant region was changed to SKSC and M14 or M58. Meanwhile, heterogeneity was significantly reduced when the constant region was SC, similar to the case with the SKSC constant region, but heterogeneity was not significantly improved when the constant region was CS.

Generally, in order to develop antibodies as pharmaceuticals, in addition to having low heterogeneity, they are also expected to have high stability in order to prepare stable formulations. Therefore, as a method for evaluating stability, differential scanning calorimetry (DSC) is used to measure the thermal denaturation intermediate temperature (Tm value) (VP-DSC, manufactured by Microcal). The thermal denaturation intermediate temperature (Tm value) is an indicator of stability. In order to prepare stable formulations as pharmaceuticals, a high thermal denaturation intermediate temperature (Tm value) is desired (J. Pharm. Sci. 2008 Apr; 97(4): 1414-26). WT-IgG1, WT-IgG2, WT-SC, WT-CS, WT-SKSC, and WT-M58 were dialyzed against a solution of 20 mM sodium acetate, 150 mM NaCl, pH 6.0 (EasySEP; TOMY). DSC measurements were performed at a protein concentration of approximately 0.1 mg/mL at a heating rate of 1°C/min in the range of 40°C to 100°C. The resulting DSC denaturation curve is shown in FIG43 , and the Tm value of the Fab portion is shown in Table 15 below.

[Table 15]

The Tm values of WT-IgG1 and WT-IgG2 are roughly equivalent, both around 94°C (the Tm value of IgG2 is about 1°C lower), while the Tm values of WT-SC and WT-CS are both approximately 86°C, which is significantly lower than those of WT-IgG1 and WT-IgG2. On the other hand, the Tm values of WT-M58 and WT-SKSC are both approximately 94°C, roughly equivalent to those of WT-IgG1 and WT-IgG2. Since the stability of WT-SC and WT-CS is significantly lower than that of IgG2, WT-SKSC and WT-M58, in which the cysteine in the CH1 domain is also substituted with serine, are considered preferable for developing antibodies as pharmaceuticals. The Tm values of WT-SC and WT-CS are significantly lower than those of IgG2, which is believed to be due to the fact that WT-SC and WT-CS differ from IgG2 in terms of disulfide bond conformation.

Comparing the DSC denaturation curves, the denaturation peaks of the Fab portion of WT-IgG1, WT-SKSC, and WT-M58 were narrow and single. In contrast, the denaturation peaks of the Fab portion of WT-SC and WT-CS were broad, and a shoulder peak was confirmed on the low-temperature side of the denaturation peak of the Fab portion of WT-IgG2. It is believed that when the DSC denaturation peak is a single component, it usually shows a narrow denaturation peak, but when multiple components with different Tm values are present (i.e., heterogeneity) are present, the denaturation peak becomes broad. In other words, the above results indicate that multiple components may be present in WT-IgG2, WT-SC, and WT-CS, and therefore the heterogeneity of native IgG2 of WT-SC and WT-CS has not been sufficiently reduced. Therefore, it is believed that the heterogeneity of wild-type IgG2 is related not only to the cysteines in the hinge region, but also to the cysteines present in the CH1 domain. In order to reduce the heterogeneity observed by DSC, it is necessary to modify not only the cysteines in the hinge region but also the cysteines in the CH1 domain. As described above, by modifying not only the cysteine residues in the hinge region but also the cysteine residues in the CH1 domain, it was possible for the first time to achieve stability equivalent to that of wild-type IgG2.

Based on these results, it is believed that the constant regions SC and CS, which only replace cysteine in the hinge region with serine, are insufficient from the perspective of heterogeneity and stability as constant regions for reducing heterogeneity from the IgG2 hinge region. It was discovered that in addition to replacing cysteine in the hinge region with serine, the cysteine at EU numbering position 131 in the CH1 domain was also replaced with serine, thereby significantly reducing heterogeneity while maintaining stability equivalent to IgG2 for the first time. Such constant regions include the aforementioned M14, M31, M58, and M73. In particular, M58 and M73 have improved plasma retention, high stability, and reduced heterogeneity, and are therefore considered very useful as constant regions for antibody drugs.

[Example 19] Preparation of a fully humanized anti-IL-6 receptor antibody with improved PK/PD

To prepare a fully humanized anti-IL-6 receptor antibody with improved PK/PD, TOCILIZUMAB (H chain, WT-IgG1 (SEQ ID NO: 15); L chain, WT (SEQ ID NO: 105)) was modified to produce the following molecules.

To improve the ka of F2H-IgG1, the affinity-enhancing modifications described in Example 2 were made: tryptophan at position 35 was substituted with valine, tyrosine at position 50 was substituted with phenylalanine, and serine at position 62 was substituted with threonine. Furthermore, to reduce the pI without increasing the risk of immunogenicity, tyrosine at position 102 was substituted with valine, glutamine at position 105 was substituted with glutamic acid, and isoleucine at position 107 was substituted with threonine. Furthermore, the constant region was converted from IgG1 to M83, creating VH5-M83 (SEQ ID NO: 139 (amino acid sequence)). To improve the ka of L39, VL5-κ (SEQ ID NO: 181 (amino acid sequence)) was created, in which glutamic acid at position 27 was substituted with glutamine. Furthermore, modified versions of tocilizumab were prepared by combining multiple mutations in the variable and constant regions of tocilizumab discovered in the above examples, as well as newly discovered mutations. Following various screenings, the following fully humanized IL-6 receptor antibodies were discovered: Fv3-M73 (H chain VH4-M73 SEQ ID NO: 182; L chain VL1-κ SEQ ID NO: 183), Fv4-M73 (H chain VH3-M73 SEQ ID NO: 180; L chain VL3-κ SEQ ID NO: 181), and Fv5-M83 (H chain VH5-M83 SEQ ID NO: 139; L chain VL5-κ SEQ ID NO: 138).

The affinity of the prepared Fv3-M73, Fv4-M73, and Fv5-M83 for the IL-6 receptor was compared with that of TOCILIZUMAB (see Reference Example for the method). The affinity of the above antibodies for the IL-6 receptor was measured, and the results are shown in Table 16. In addition, the BaF/gp130 neutralization activity of TOCILIZUMAB and a control (a known high-affinity anti-IL-6 receptor antibody from Reference Example, VQ8F11-21 hIgG1 from US 2007/0280945) was compared (see Reference Example for the method). The biological activity of these antibodies against BaF/gp130 was measured, and the results are shown in Figure 44 (final IL-6 concentration of 300 ng/mL: TOCILIZUMAB, control, Fv5-M83) and Figure 45 (final IL-6 concentration of 30 ng/mL: TOCILIZUMAB, Fv3-M73, Fv4-M73). As shown in Table 16, Fv3-M73 and Fv4-M73 had approximately 2-3 times stronger affinity than tocilizumab, while Fv5-M83 exhibited approximately 100 times stronger affinity than tocilizumab (because the affinity of Fv5-M83 was difficult to measure, affinity was measured using Fv5-IgG1, whose constant region is IgG1, as it is believed that the constant region generally has no effect on affinity). As shown in Figure 45, Fv3-M73 and Fv4-M73 exhibited slightly stronger activity than tocilizumab. As shown in Figure 44, Fv5-M83 exhibited extremely strong activity, more than 100 times stronger than tocilizumab at a 50% inhibitory concentration. Furthermore, Fv5-M83 exhibited approximately 10 times higher neutralizing activity at a 50% inhibitory concentration than the control (a known high-affinity anti-IL-6 receptor antibody).

[Table 16]

The isoelectric points of tocilizumab, a control, Fv3-M73, Fv4-M73, and Fv5-M83 were measured by isoelectric electrophoresis using methods known to those skilled in the art. The results showed that the isoelectric point of tocilizumab was approximately 9.3, the isoelectric point of the control was approximately 8.4-8.5, the isoelectric point of Fv3-M73 was approximately 5.7-5.8, the isoelectric point of Fv4-M73 was approximately 5.6-5.7, and the isoelectric point of Fv5-M83 was approximately 5.4-5.5. The isoelectric points of these antibodies were significantly lower than those of tocilizumab and the control. Furthermore, when the theoretical isoelectric points of the variable regions VH/VL were calculated using GENETYX (GENETYX CORPORATION), the theoretical isoelectric points of tocilizumab were 9.20, those of the control were 7.79, those of Fv3-M73 were 5.49, those of Fv4-M73 were 5.01, and those of Fv5-M83 were 4.27. These theoretical isoelectric points were significantly lower than those of tocilizumab and the control. This suggests that the plasma retention of Fv3-M73, Fv4-M73, and Fv5-M83 was improved compared to those of tocilizumab and the control.

T cell epitopes present in the variable region sequences of TOCILIZUMAB, Fv3-M73, Fv4-M73, and Fv5-M83 were analyzed using TEPITOPE (Methods. 2004 Dec; 34(4): 468-75). The results showed that while multiple sequences of TOCILIZUMAB were predicted to contain T cell epitopes that bind to HLA, the number of sequences predicted to bind to T cell epitopes was significantly reduced in Fv3-M73, Fv4-M73, and Fv5-M83. Furthermore, Fv3-M73, Fv4-M73, and Fv5-M83 have no residual mouse sequences in their frameworks and are fully humanized. This suggests that the immunogenicity risk of Fv3-M73, Fv4-M73, and Fv5-M83 is likely to be significantly reduced compared to TOCILIZUMAB.

[Example 20] PK/PD test of fully humanized anti-IL-6 receptor antibody in monkeys

Tocilizumab, a control, Fv3-M73, Fv4-M73, and Fv5-M83 were administered a single intravenous dose of 1 mg/kg to cynomolgus monkeys, and changes in plasma concentrations were evaluated (see the Reference Example for the method). Changes in plasma concentrations of Tocilizumab, Fv3-M73, Fv4-M73, and Fv5-M83 after intravenous administration are shown in Figure 46. The results showed that compared to Tocilizumab and the control, Fv3-M73, Fv4-M73, and Fv5-M83 significantly improved plasma retention in cynomolgus monkeys. In particular, Fv3-M73 and Fv4-M73 significantly improved plasma retention compared to Tocilizumab.

In order to evaluate the efficacy of each antibody in neutralizing the membrane-type cynomolgus monkey IL-6 receptor, cynomolgus monkey IL-6 was subcutaneously administered to the lower back at 5 μg/kg for consecutive days from the 6th to the 18th day after antibody administration (the 3rd to the 10th day after TOCILIZUMAB administration), and the CRP concentration of each individual was measured 24 hours later (see the reference example for the method). The changes in CRP concentration after administration of each antibody are shown in Figure 47. In order to evaluate the efficacy of each antibody in neutralizing the soluble cynomolgus monkey IL-6 receptor, the concentration of unbound soluble cynomolgus monkey IL-6 receptor in cynomolgus monkey plasma was measured, and the neutralization rate of the soluble IL-6 receptor was calculated (see the reference example for the method). The changes in the neutralization rate of the soluble IL-6 receptor after administration of each antibody are shown in Figure 48.

Compared to tocilizumab and a control (a known high-affinity anti-IL-6 receptor antibody), Fv3-M73, Fv4-M73, and Fv5-M83 all neutralized the membrane-bound cynomolgus monkey IL-6 receptor more persistently, suppressing the increase in CRP over a long period of time. Furthermore, compared to tocilizumab and the control, Fv3-M73, Fv4-M73, and Fv5-M83 all neutralized the soluble cynomolgus monkey IL-6 receptor more persistently, suppressing the increase in unbound soluble cynomolgus monkey IL-6 receptor over a long period of time. Thus, it was found that Fv3-M73, Fv4-M73, and Fv5-M83 all outperformed tocilizumab and the control in terms of the persistence of neutralization of the membrane-bound IL-6 receptor and the soluble IL-6 receptor. Among them, the persistence of neutralization by Fv3-M73 and Fv4-M73 was extremely excellent. On the other hand, Fv5-M83 suppressed CRP and unbound soluble cynomolgus monkey IL-6 receptor at lower levels compared to Fv3-M73 and Fv4-M73. This suggests that Fv5-M83 more potently neutralizes membrane-bound and soluble IL-6 receptors than Fv3-M73, Fv4-M73, and a control (a known high-affinity anti-IL-6 receptor antibody). This is believed to be due to the stronger affinity of Fv5-M83 for the IL-6 receptor compared to the control, as well as the strong biological activity of Fv5-M83 in BaF/gp130, which is reflected in cynomolgus monkeys.

These studies demonstrate that Fv3-M73 and Fv4-M73 exhibit significantly superior duration of action as anti-IL-6 receptor neutralizing antibodies compared to tocilizumab and controls, allowing for significantly reduced dosing frequency and dosage. Furthermore, Fv5-M83 exhibits exceptionally high potency and duration of action as an anti-IL-6 receptor neutralizing antibody. Therefore, it is believed that Fv3-M73, Fv4-M73, and Fv5-M83 can be used as pharmaceuticals as IL-6 antagonists.

[Reference example]

Preparation of recombinant soluble cynomolgus monkey IL-6 receptor (cIL-6R)

OligoDNA primers were prepared based on the published rhesus macaque IL-6 receptor gene sequence (Birney et al., Ensembl 2006, Nucleic Acids Res. 2006 Jan 1;34 (Database issue):D556-61). Using cDNA prepared from cynomolgus macaque pancreas as a template, these primers were used to generate a DNA fragment encoding the full-length cynomolgus macaque IL-6 receptor gene by PCR. The resulting DNA fragment was inserted into an animal cell expression vector, and this vector was used to generate a CHO constant expression strain (cynomolgus macaque sIL-6R-producing CHO cell line). The culture medium of the cynomolgus macaque sIL-6R-producing CHO cell line was purified using a HisTrap column (GE Healthcare Bioscience), then concentrated using an Amicon Ultra-15 Ultracel-10k (Millipore), and further purified using a Superdex 200 pg 16/60 gel filtration column (GE Healthcare Bioscience) to obtain a final purified product of soluble cynomolgus macaque IL-6 receptor (hereinafter referred to as cIL-6R).

Production of recombinant cynomolgus monkey IL-6 (cIL-6)

Cynomolgus monkey IL-6 was prepared as follows. A nucleotide sequence encoding 212 amino acids registered in SWISSPROT Accession No. P79341 was prepared, cloned into an animal cell expression vector, and the resulting vector was introduced into CHO cells to create a constant expression cell line (CHO cells producing cynomolgus monkey IL-6). The culture medium of the CHO cells producing cynomolgus monkey IL-6 was purified using an SP-Sepharose/FF column (GE Healthcare Bioscience), then concentrated using an Amicon Ultra-15 Ultracel-5k (Millipore), further purified using a Superdex 75pg 26/60 gel filtration column (GE Healthcare Bioscience), and concentrated using an Amicon Ultra-15 Ultracel-5k (Millipore) to obtain the final pure product of cynomolgus monkey IL-6 (hereinafter referred to as cIL-6).

Preparation of known high-affinity anti-IL-6 receptor antibodies

To express the well-known high-affinity anti-IL-6 receptor antibody VQ8F11-21 hIgG1 described in US 2007/0280945A1 (US 2007/0280945A1, H chain amino acid sequence, SEQ ID NO: 19; L chain amino acid sequence, SEQ ID NO: 27), an animal cell expression vector was constructed. The antibody variable region was produced by PCR combined with synthetic oligoDNA (assembly PCR), while IgG1 was used for the constant region. The antibody variable region and constant region were combined using assembly PCR and then inserted into an animal cell expression vector to produce the desired H chain and L chain expression vectors. The nucleotide sequence of the resulting expression vector was determined according to methods known to those skilled in the art, and expression and purification were performed using the produced expression vector. Expression and purification were performed according to the methods described in Example 1 to obtain a high-affinity anti-IL-6 receptor antibody (hereinafter referred to as a control).

Evaluation of IL-6 receptor binding using Biacore

Biacore T100 (GE Healthcare) was used to analyze the rate of antigen-antibody reaction. An appropriate amount of anti-IgG (γ-chain specific) F(ab') ₂ was immobilized on the sensor chip by amine coupling. Next, the target antibody was bound at pH 7.4, and then the IL-6 receptor, i.e., SR344, adjusted to various concentrations, was flowed as an analyte at pH 7.4 to measure the interaction between the antibody and SR344. All measurements were performed at 37°C. The kinetic parameters, i.e., the binding rate constant _ka (1/Ms) and the dissociation rate constant _kd (1/s), were calculated from the sensorgrams obtained in the measurement, and _KD (M) was calculated based on the values. Each parameter was calculated using Biacore T100 evaluation software (GE Healthcare).

The plasma antibody concentration, CRP concentration, and unbound soluble IL-6 receptor were determined by monkey PK/PD assay. Body concentration

The concentration in cynomolgus monkey plasma was measured by ELISA using a method known to those skilled in the art. CRP concentration was measured using Cias R CRP (Kanto Chemical Co., Ltd.) using an automatic analyzer (TBA-120FR, Toshiba Medical Systems Co., Ltd.).

The concentration of unbound soluble cynomolgus monkey IL-6 receptor in cynomolgus monkey plasma was determined as follows. 30 μL of cynomolgus monkey plasma was added to an appropriate amount of rProtein A Sepharose FastFlow resin (GE Healthcare) dried in a 0.22 μm filter cup (Millipore) so that all IgG antibodies (cynomolgus monkey IgG, anti-human IL-6 receptor antibody, anti-human IL-6 receptor antibody-soluble cynomolgus monkey IL-6 receptor complex) present in the plasma were adsorbed on protein A. Afterwards, the solution was spun down in a high-speed centrifuge to recover the solution that passed through. Since the solution that passed through did not contain the anti-human IL-6 receptor antibody-soluble cynomolgus monkey IL-6 receptor complex bound to protein A, the concentration of unbound soluble IL-6 receptor was determined by measuring the concentration of soluble cynomolgus monkey IL-6 receptor in the solution that passed through protein A. The concentration of soluble cynomolgus monkey IL-6 receptor was determined using the previously prepared soluble cynomolgus monkey IL-6 receptor (cIL-6R) as a standard using methods known to those skilled in the art for measuring human IL-6 receptor concentration. The neutralization rate of soluble IL-6 receptor was calculated using the following formula.

(Unbound soluble IL-6 receptor concentration after antibody administration ÷ soluble IL-6 receptor concentration before antibody administration) × 100

[Example 21]

(1) Preparation of point mutation gene of humanized H0L0 antibody

Using the gene encoding the glypican 3 antibody containing the humanized GC33 antibody CDR disclosed in WO2006/046751 as the starting material, various point mutation genes were made. Oligo DNA designed according to the sequence of the positive and reverse strands containing the modification site was synthesized. A commercially available QuikChange site-directed mutagenesis kit (Stratagene) was used to make multiple point mutation genes. The point mutation gene was made according to the following conditions and by PCR. The reaction mixture consisting of 10 ng template plasmid, 10 pmol of synthetic oligo DNA of the positive and reverse strands, 10×Buffer, dNTP mix and Pfu turbo DNA polymerase added to the kit was heated at 95°C for 30 seconds, followed by 18 PCR reaction cycles consisting of 95°C×30 seconds, 55°C×1 minute, and 68°C×4 minutes. DpnI added to the kit was added to the reaction mixture, followed by a restriction digest reaction caused by the restriction enzyme at 37°C for 1 hour. This reaction solution was used to transform DH5α competent cells (TOYOBO), resulting in transformants. The nucleotide sequence of the plasmid DNA isolated from the transformants was determined, and the point mutation gene, confirmed to have introduced a point mutation, was cloned into an expression vector capable of expressing the inserted gene in animal cells. The modified gene was obtained by modification to have the following structure.

Transient expression of the humanized H0L0 antibody and its point mutation-modified antibodies was performed using polyethyleneimine (Polysciences Inc.). HEK293 cells, detached with trypsin-EDTA (Invitrogen), were seeded in a 10 ^cm² culture dish to a density of 6× ^10⁶ cells/10 mL. The next day, 690 μL of SFMII medium and 20.8 μg of polyethyleneimine were added to 4.6 μg of H-chain expression plasmid DNA and 9.2 μg of L-chain expression plasmid DNA. After mixing, the mixture was allowed to stand at room temperature for 10 minutes. The entire mixture was added dropwise to the culture dish seeded with HEK293 cells as described above. After approximately 72 hours, the culture supernatant was recovered, and the expressed humanized H0L0 antibody and its point mutation-modified antibodies were purified from the recovered culture supernatant using rProteinA sepharose™ Fast Flow (GE Healthcare) according to the manufacturer's instructions.

(1-1) Changing the Tm value of humanized H0L0 antibody

The thermal denaturation intermediate temperature (Tm) is determined by the apex of the denaturation peak in the resulting thermal map (Cp vs. T plot) after heating the sample solution at a programmed constant heating rate. The sample solution for DSC measurement was prepared as follows, and the Tm value of the humanized H0L0 antibody was determined. First, a 50-100 μg amount of antibody solution enclosed in a dialysis membrane was dialyzed overnight using a 20 mol/l sodium acetate buffer solution (pH 6.0) containing 150 mmol/l sodium chloride as the dialysis external fluid. A sample solution with an antibody concentration of 50-100 μg/mL was then prepared using the dialysis external fluid, and this sample solution was used as the sample solution for DSC measurement.

To conduct this experiment, it is preferred to use an appropriate DSC apparatus, such as DSC-II (Calorimetry Sciences Corporation). The sample solution and reference solution (dialysis external solution) prepared using the above method are fully degassed, and then each test specimen is sealed in a calorimeter box and fully thermally balanced at 40°C. Next, a DSC scan is performed in the range of 40°C to 100°C at a scanning speed of approximately 1K/min. The measurement results are represented by the vertex of the denaturation peak as a function of temperature. The peak of the Fab domain is specified with reference to non-patent literature (Rodolfo et al., Immunology Letters (1999), 47-52), and the thermal denaturation intermediate temperature of the humanized H0L0 antibody is calculated. As a specific example, Figure 49 illustrates a graph obtained by DSC (differential scanning calorimeter) measurement of the Hspu2.2Lspu2.2 (Hu2.2Lu2.2) antibody.

The Tm value of the humanized H0L0 antibody, which comprises the H chain represented by SEQ ID NO: 195 and the L chain represented by SEQ ID NO: 201, calculated using the above method, was 76.6°C. In contrast, the Tm values of Synagis and Herceptin, exemplified as existing antibodies, were measured to be 85.4°C and 81.8°C, respectively. This indicates that the Tm value of the humanized H0L0 antibody is lower than those of existing antibodies.

To improve the Tm value of the humanized H0L0 antibody, modified versions of the humanized H0L0 antibody were created. H15 (SEQ ID NO: 196) was created by modifying the FR2 of the H chain of the H0L0 antibody shown in SEQ ID NO: 195 from the VH1b subclass to V37I, A40P, M48I, and L51I of the VH4 subclass. This improved its Tm value to 79.1°C. L4 (SEQ ID NO: 202) was created by modifying the FR2 of the L chain of the H0L0 antibody shown in SEQ ID NO: 201 from VK2 to L42Q, S48A, and Q50R of the VK3 subclass, and by replacing V2 of FR1 with V2I of the germline sequence I. This improved its Tm value to 77.2°C. The combination of these two modifications improved the Tm value of the H15L4 antibody to 80.5°C.

(1-2) Changing the pI value of humanized H0L0 antibody

By reducing the pI value of an antibody, its blood half-life is prolonged. Conversely, by increasing the pI value of an antibody, its tissue mobility is improved. It is unclear whether increasing or decreasing the pI value of an antibody that exhibits a cancer therapeutic effect enhances its tumor suppression effect. Therefore, we prepared modified humanized H0L0 antibodies with reduced and increased pI values, and compared their antitumor effects to determine whether either modification resulted in a greater tumor suppression effect.

The pI value of each antibody was calculated by isoelectric electrophoresis analysis. The electrophoresis was performed as follows: Using a Phastsystem Cassette (made by American Bioscience), a Phast-Gel Dry IEF (American Bioscience) gel was swollen with a swelling solution having the following composition for approximately 60 minutes.

(a) Composition of high pI swelling fluid:

1.5 mL 10% glycerol

100 μL IEF with Pharmalyte 8-10.5 (Amercham Bioscience)

(b) Composition of low pI swelling fluid:

1.5 mL purified water

20 μL IEF with Pharmalyte 8-10.5 (Amercham Bioscience)

80μL IEF with Pharmalyte 5-8 (AmerchamBioscience)

About 0.5 μg of antibody was added to the swollen gel, and isoelectric electrophoresis was performed using the Phast System (American Bioscience) controlled by the following program. The sample was added to the gel in step 2 of the following program. For pI markers, a pI calibration kit (American Bioscience) was used.

Step 1: 2000V, 2.5mA, 3.5W, 15°C, 75Vh

Step 2: 200V, 2.5mA, 3.5W, 15°C, 15Vh

Step 3: 2000V, 2.5mA, 3.5W, 15°C, 410Vh

After electrophoresis, the gel was fixed with 20% TCA and then silver-stained using a silver staining kit and protein (Amercham Bioscience) according to the kit's instructions. After staining, the isoelectric point of each antibody in the test sample was calculated based on the known isoelectric point of the pI marker. The electrophoretic images of high- and low-pI isoelectric point electrophoresis are shown in Figures 50 and 51, respectively.

(a) Modification to increase pI value

H15 was further modified with Q43K, D52N, and Q107R to produce Hspu2.2 (Hu2.2) (SEQ ID NO: 200). Furthermore, L4 was modified with E17Q, Q27R, Q105R, and S25A (S25 of CDR2 was substituted with A, which is high frequency in the germline) to produce Lspu2.2 (Lu2.2) (SEQ ID NO: 206). The antibody comprising Hspu2.2 (Hu2.2) and Lspu2.2 (Lu2.2), i.e., the Hspu2.2Lspu2.2 (Hu2.2Lu2.2) antibody, had a Tm value of 76.8°C and a pI value of 9.6. Since the pI value of the H0L0 antibody was 8.9, the pI value of the Hspu2.2Lspu2.2 (Hu2.2Lu2.2) antibody was increased by 0.7.

(b) Modification to reduce pI value

H15 was further modified with K19T, Q43E, K63S, K65Q, and G66D to produce Hspd1.8 (Hd1.8) (SEQ ID NO: 199). L4 was modified with Q27E, and the sequence 79-84 of FR3 of L4, i.e., KISRVE, was modified with TISSLQ. Similarly, Lspu2.2 (Lu2.2) was modified with S25A to produce Lspd1.6 (Ld1.6) (SEQ ID NO: 205). The antibody comprising Hspd1.8 (Hd1.8) and Lspd1.6 (Ld1.6), i.e., the Hspd1.8Lspd1.6 (Hd1.8Ld1.6) antibody, was measured to have a Tm value of 72.6°C and a pI value of 7.4. Since the pI value of the H0L0 antibody is 8.9, the pI value of the Hspd1.8Lspd1.6 (Hd1.8Ld1.6) antibody is reduced by 1.5.

(2) Evaluation of the binding activity of H0L0 antibody point mutation-modified antibodies by competitive ELISA

The purified H0L0 antibody and its point mutation modified antibody in (1) were evaluated by competitive ELISA. 100 μL of soluble GPC3 core polypeptide (SEQ ID NO: 207) prepared at 1 μg/mL was added to each well of a 96-well plate. The plate was left to stand at 4°C overnight to immobilize the soluble GPC3 core polypeptide on the plate. The soluble GPC3 core polypeptide immobilized on the plate was washed three times with washing buffer using Skan WASHER 400 (Molecular Devices), and 200 μL of blocking buffer was added and blocked at 4°C for more than one night. Next, the plate with the immobilized and blocked soluble GPC3 core polypeptide was washed three times with washing buffer using Skan WASHER 400. Thereafter, 100 μL of a mixture of various concentrations of H0L0 antibody or its point mutation modified antibody and a final concentration of 0.3 μg/mL of biotinylated H0L0 antibody was added to each well of the plate. Biotinylation of the H0L0 antibody was performed using a biotin labeling kit (Roche) according to the kit's instructions. After the plate was allowed to stand at room temperature for 1 hour, it was washed five times with wash buffer using a Skan WASHER 400 (Molecular Devices). 100 μL of goat anti-streptavidin alkaline phosphatase (ZYMED) diluted to 20,000-fold in matrix buffer was added to each well. The plate was allowed to stand at room temperature for 1 hour, and then washed five times with wash buffer using a Skan WASHER 400. Phosphatase substrate (Sigma) was prepared to 1 mg/mL using matrix buffer, and then 100 μL was added to each well and allowed to stand for 1 hour. The absorbance of the reaction solution in each well at 405 nm was measured using a Benchmark Plus (BIO-RAD) using a control absorbance at 655 nm.

As shown in Figure 52 , the antigen-binding activity of the H15L4 antibody was nearly equivalent to that of the modified H0L0 antibody. Furthermore, as shown in Figure 53 , the antigen-binding activity of the Hspu2.2Lspu2.2 (Hu2.2Lu2.2) antibody was nearly equivalent to that of the modified H0L0 antibody. Furthermore, as shown in Figure 54 , the antigen-binding activity of the Hspd1.8Lspd1.6 (Hd1.8Ld1.6) antibody was nearly equivalent to that of the modified H0L0 antibody.

[Reference Example 22] Disruption of the fucose transporter gene in CHO cells

(1) Construction of targeting vector

(1-1) Preparation of KO1 vector

Using pcDNA3.1/Hygro (Invitrogen) and primers Hyg5-BH and Hyg3-NT, a BamH I site and a TGCGC sequence were added to the 5' side of the start codon of the hygromycin resistance gene (Hygr) by PCR to form a sequence identical to the 5' side of the start codon of the fucose transporter gene. A Not I site was added to the 3' side of the region including up to the SV40 polyA addition signal to select Hygr.

Forward primer

Hyg5-BH: 5’-GGATCCTGCGCATGAAAAAGCCTGAACTCACC-3’ (SEQ ID NO: 208)

Reverse primer

Hyg3-NT: 5’-GCGGCCGCCTATTCCTTTGCCCTCGGACG-3’ (SEQ ID NO: 209)

The fucose transporter targeting vector ver. 1 (hereinafter referred to as the KO1 vector) was constructed by inserting the fucose transporter fragments 5' (SmaI from position 2,780 to BamHI from position 4,232 of the nucleotide sequence shown in SEQ ID NO: 210), 3' (SacI from position 4,284 to position 10,934), and the Hygr fragment into the pMC1DT-A vector (Yagi T, Proc. Natl. Acad. Sci. USA (1990) 87, 9918-22). This vector is characterized by the lack of a promoter within Hygr, so upon homologous recombination, Hygr is expressed via the fucose transporter promoter. However, when only a single copy of the vector is introduced into cells via homologous recombination, the expression of Hygr is insufficient to confer hygromycin B resistance. It should be noted that the KO1 vector was digested with NotI prior to introduction into cells. It is believed that the introduction of the KO1 vector caused the deletion of 41 base pairs of exon 1 including the start codon of the fucose transporter, resulting in its loss of function.

(1-2) Preparation of pBSK-pgk-1-Hygr

The promoter of the mouse pgk-1 gene was excised from the pKJ2 vector (Popo H, Biochemical Genetics (1990) 28, 299-308) using EcoRI-PstI and cloned into the EcoRI-PstI site of pBluescript (Stratagene) to create pBSK-pgk-1. Hygr was isolated by PCR using pcDNA3.1/Hygro and primers Hyg5-AV and Hyg3-BH. An EcoT22I site and a Kozak sequence were added to the 5' side of Hygr, and a BamHI site was added to the 3' side of the region encompassing the SV40 polyA addition signal.

Forward primer

Hyg5-AV: 5’-ATGCATGCCACCATGAAAAAGGCCTGAACTCACC-3’ (SEQ ID NO: 211)

Reverse primer

Hyg3-BH: 5’-GGATCCCAGGCTTTACACTTTATGCTTC-3’ (SEQ ID NO: 212)

This Hygr (EcoT22 I-BamHI) fragment was inserted into the PstI-BamHI site of pBSK-pgk-1 to construct pBSK-pgk-1-Hygr.

(1-3) Preparation of KO2 vector

The fucose transporter targeting vector version 2 (hereinafter referred to as the KO2 vector) was constructed by inserting the fucose transporter fragments 5' (Sma I from position 2,780 to BamH I from position 4,232 of the nucleotide sequence shown in SEQ ID NO: 210) and 3' (Sac I from position 4,284 to position 10,934) into the pMC1DT-A vector. Unlike the KO1 vector, the KO2 vector incorporates the pgk-1 gene promoter within the Hygr fragment, allowing hygromycin B resistance to be conferred even when only a single copy of the vector is introduced into cells via homologous recombination. The KO2 vector was digested with Not I prior to introduction into cells. It is believed that the introduction of the KO2 vector deletes the 46 base pairs of exon 1, including the start codon, leading to functional loss of the fucose transporter.

(1-4) Preparation of pBSK-pgk-1-Puror

The pPUR vector (BD Biosciences) was digested with Pst I and Bam HI, and the excised fragment (Puror) was inserted into the Pst I-Bam HI site of pBSK-pgk-1 to construct pBSK-pgk-1-Puror.

(1-5) Preparation of KO3 vector

The fucose transporter targeting vector version 3 (hereinafter referred to as the KO3 vector) was constructed by inserting the fucose transporter fragments on the 5' side (Sma I from position 2,780 to BamH I from position 4,232 of the nucleotide sequence shown in SEQ ID NO: 210), Sac I from position 4,284 to position 10,934), and the pgk-1-Puror fragment into the pMC1DT-A vector. It should be noted that a sequence bound by the screening primer shown below was pre-added to the 3' end of pgk-1-Puror. The KO3 vector was digested with Not I and then introduced into cells. It is believed that the introduction of the KO3 vector deletes the 46 base pairs of exon 1, including the start codon, causing the fucose transporter to lose its function.

Reverse primer

RSGR-A 5’-GCTGTCTGGAGTACTGTGCATCTGC-3’(SEQ ID NO:213)

Using the above three targeting vectors, we attempted to knock out the fucose transporter gene.

(2) Introducing the vector into CHO cells

HT Supplement (100x) (Invitrogen) and penicillin-streptomycin (Invitrogen) were added to CHO-S-SFMII HT- (Invitrogen) in an amount of 1/100 of the capacity of CHO-S-SFMII HT-. This was used as a culture medium (hereinafter referred to as SFMII (+)) to passage the DXB11 strain of CHO cells, and the culture after the gene was introduced was also carried out in this SFMII (+). 8 × 10 ⁶ CHO cells were suspended in 0.8 mL Dulbecco's phosphate buffer (hereinafter referred to as PBS. Invitrogen). 30 μg of targeting vector was added to the cell suspension and the cell suspension was moved to a GenePulser Cuvette (4 mm) (Bio-Rad). After standing on ice for 10 minutes, the vector was introduced into the cell by electroporation using GENE-PULSER II (Bio-Rad) at 1.5 kV and 25 μFD. After vector introduction, cells were suspended in 200 mL of SFMII(+) medium and seeded into 20 96-well flat-bottom plates (Iwaki) at 100 μL/well. The plates were incubated at 37°C in a _CO2 incubator for 24 hours before adding the drug.

(3) The first stage of knockout

KO1 vector or KO2 vector was introduced into CHO cells, and selection was performed 24 hours after vector introduction using hygromycin B (Invitrogen). Hygromycin B was dissolved in SFMII(+) to a concentration of 0.3 mg/mL and added at 100 μL/well.

(4) Screening of homologous recombinants by PCR

(4-1) Preparation of PCR samples

Homologous recombinants were screened using PCR. The CHO cells used in the screening were cultured in 96-well flat-bottom plates. After removing the culture supernatant, 50 μL/well of cell lysis buffer was added and heated at 55°C for 2 hours, followed by heating at 95°C for 15 minutes to inactivate proteinase K and form a PCR template. The cell lysis buffer in each well consisted of 5 μL of 10X LA buffer II (added to Takara LATaq), 2.5 μL of 10% NP-40 (Roche), 4 μL of proteinase K (20 mg/mL, Takara), and 38.5 μL of distilled water (Nacalai Tesque).

(4-2) PCR conditions

The PCR reaction mixture consisted of 1 μL of the above-mentioned PCR sample, 5 μL of 10× LA buffer II, 5 μL of MgCl2 (25 mM), 5 μL of dNTPs (2.5 mM), 2 μL of primers (10 μM each), 0.5 μL of LA Taq (5 IU/μL), and 29.5 μL of distilled water (50 μL total). TP-F4 and THygro-R1 were used as PCR primers for screening cells transfected with the KO1 vector; TP-F4 and THygro-F1 were used for screening cells transfected with the KO2 vector.

PCR conditions for cells transfected with the KO1 vector were: preheating at 95°C for 1 minute, followed by 40 amplification cycles (95°C for 30 seconds, 60°C for 30 seconds, and 70°C for 2 minutes), and then heating at 72°C for 7 minutes. For screening of cells transfected with the KO2 vector, PCR conditions were: preheating at 95°C for 1 minute, followed by 40 amplification cycles (95°C for 30 seconds and 70°C for 3 minutes), and then heating at 70°C for 7 minutes.

The primers are as follows. Approximately 1.6 kb of DNA was amplified in cell samples undergoing homologous recombination mediated by the KO1 vector; approximately 2.0 kb of DNA was amplified in cell samples undergoing homologous recombination mediated by the KO2 vector. Primer TP-F4 was designed to target the fucose transporter genomic region outside the vector and 5' to the genomic region. THygro-F1 and THygro-R1 were designed to target the Hygr region within the vector.

Forward primers (KO1, KO2)

TP-F4: 5’-GGAATGCAGCTTCCTCAAGGGACTCGC-3’ (SEQ ID NO: 214)

Reverse primer (KO1)

THygro-R1: 5’-TGCATCAGGTCGGAGACGCTGTCGAAC-3’ (SEQ ID NO: 215)

Reverse primer (KO2)

THygro-F1: 5’-GCACTCGTCCGAGGGCAAAGGAATAGC-3’ (SEQ ID NO: 216)

(5) PCR screening results

Of the 918 cells transfected with the KO1 vector, only one was considered a homologous recombinant (homologous recombination efficiency approximately 0.1%). Furthermore, of the 537 cells transfected with the KO2 vector, only 17 were considered homologous recombinants (homologous recombination efficiency approximately 3.2%).

(6) DNA blot analysis

Recombinant cells were further confirmed by Southern blotting. 10 μg of genomic DNA was prepared from the cultured cells according to conventional methods and confirmed by Southern blotting. A 387 bp probe was generated by PCR from the region between nucleotide positions 2,113 and 2,500 of the nucleotide sequence of SEQ ID NO: 210 using the following two primers. This probe was used for Southern blotting. The genomic DNA was digested with Bgl II.

Forward primer

Bgl-F: 5'-TGTGCTGGGAATTGAACCCAGGAC-3' (SEQ ID NO: 217) reverse primer

Bgl-R: 5’-CTACTTGTCTGTGCTTCTTCC-3’ (SEQ ID NO: 218)

Bgl II cleavage revealed an approximately 3.0 kb band from the chromosome harboring the fucose transporter, an approximately 4.6 kb band from the chromosome harboring homologous recombination mediated by the KO1 vector, and an approximately 5.0 kb band from the chromosome harboring homologous recombination mediated by the KO2 vector. One cell type harboring homologous recombination mediated by the KO1 vector and seven cells harboring homologous recombination mediated by the KO2 vector were used in the experiment. The only cell obtained using the KO1 vector was named 5C1. However, subsequent analysis revealed that 5C1 consisted of multiple cell populations, so it was cloned by limiting dilution and used in subsequent experiments. Additionally, one of the cells obtained using the KO2 vector was named 6E2.

(7) The second stage of knockout

For cells that successfully underwent homologous recombination mediated by the KO1 vector and the KO2 vector, we attempted to establish a cell line with a complete deletion of the fucose transporter gene using three vectors. The combinations of vectors and cells are as follows. Method 1: KO2 vector and 5C1 cells (KO1), Method 2: KO2 vector and 6E2 cells (KO2), Method 3: KO3 vector and 6E2 cells (KO2). The vectors were introduced into each cell, and screening was started using hygromycin B and puromycin (Nacalai Tesque) 24 hours after the vector introduction. The final concentration of hygromycin B in Method 1 was 1 mg/mL, and the final concentration of hygromycin B in Method 2 was 7 mg/mL. In addition, in Method 3, hygromycin B and puromycin were added to a final concentration of 0.15 mg/mL and 8 μg/mL, respectively.

(8) Screening of homologous recombinants by PCR

The sample was prepared as follows. In the screening of method 1, two PCRs were performed to detect cells that had undergone homologous recombination mediated by the above-mentioned KO1 vector and KO2 vector. In method 2, the following PCR primers were designed. TPS-F1 was set in the region from position 3,924 to position 3,950 of the nucleotide sequence shown in SEQ ID NO: 210, and SHygro-R1 was set in the region from position 4,248 to position 4,274. These PCR primers amplified 350 bp of the fucose transporter gene region deleted by the KO2 vector. Therefore, in the PCR screening of method 2, the 350 bp of the gene region that was not amplified was considered to be a cell with a complete deletion of the fucose transporter gene. The PCR conditions were as follows: preheating at 95°C for 1 minute, 35 amplification cycles (95°C × 30 seconds, 70°C × 1 minute as one amplification cycle), and heating again at 70°C for 7 minutes.

Forward primer

TPS-F1: 5’-CTCGACTCGTCCCTATTAGGCAACAGC-3’ (SEQ ID NO: 219)

Reverse primer

SHygro-R1: 5’-TCAGAGGCAGTGGAGCCTCCAGTCAGC-3’ (SEQ ID NO: 220)

In method 3, TP-F4 was used as the forward primer and RSGR-A was used as the reverse primer. The PCR conditions were as follows: preheating at 95°C for 1 minute, followed by 35 amplification cycles (95°C × 30 seconds, 60°C × 30 seconds, and 72°C for 2 minutes as one amplification cycle), and then heating again at 72°C for 7 minutes. In the cell sample that underwent homologous recombination mediated by the KO3 vector, approximately 1.6 kb of DNA was amplified. This PCR detected cells that underwent homologous recombination mediated by the KO3 vector, and also confirmed the presence of residual homologous recombination mediated by the KO2 vector.

(9) PCR screening results

In method 1, 616 cells were analyzed, of which 18 were considered to be identical recombinants (identical recombination efficiency was 2.9%). In method 2, 524 cells were analyzed, of which 2 were considered to be identical recombinants (identical recombination efficiency was approximately 0.4%). Furthermore, in method 3, 382 cells were analyzed, of which 7 were considered to be identical recombinants (identical recombination efficiency was approximately 1.8%).

(10) DNA blot analysis

Analysis was performed using the above method. Among the cells that could be analyzed, one was found to have a complete deletion of the fucose transporter gene. While the results of PCR and Southern blotting were consistent in the first-stage knockout, they were inconsistent in the second-stage knockout.

(11) Fucose expression analysis

The expression of fucose in 26 cells identified as homologous recombinants by PCR was further analyzed. 1×10 6 cells were stained on ice for 1 hour using ¹⁰⁰ μL of PBS containing 5 μg/mL Lens culinaris Agglutinin, FITC conjugate (Vector Laboratories), 2.5% FBS, and 0.02% sodium azide (hereinafter referred to as FACS lysate). Afterwards, the cells were washed three times with FACS lysate and measured using a FACSCalibur (Becton Dickinson). Southern blot analysis showed that the expression of fucose was reduced only in the FTP-KO strain, a cell line identified as completely lacking the fucose transporter gene.

[Reference Example 23] Establishment of Antibody-Producing Cells from FTP-KO Strain and Antibodies Produced by These Cells purification

Prepare SFMII (+) medium containing hygromycin B so that the final concentration of hygromycin B in SFMII (+) medium is 1 mg/mL, and passage the fucose transporter deletion strain (FT-KO cells, clone name 3F2) obtained in Example 21. Suspend 8×10 ⁶ 3F2 cells in 0.8 mL Dulbecco's phosphate buffer. Add 25 μg of humanized glypican 3 antibody expression vector to the cell suspension, and transfer the cell suspension to a Gene Pulser Cuvette. After placing on ice for 10 minutes, use GENE-PULSER II to introduce the vector into the cells by electroporation at 1.5 kV and 25 μFD. After the vector is introduced, the cells are suspended in 40 mL SFMII (+) medium and then seeded into a 96-well flat-bottom plate (Iwaki) at 100 μL/well. The plate was incubated at 37°C in a _CO2 incubator for 24 hours, after which Geneticin (Invitrogen) was added to a final concentration of 0.5 mg/mL. The antibody production of drug-resistant cells was measured, and humanized Glypican 3 antibody-producing cell lines were established.

Reclaim the culture supernatant from the antibody expression strain and use a P-1 pump (Pharmacia) to add sample to a Hitrap rProtein A (Pharmacia) column. Use binding buffer (20mM sodium phosphate (pH 7.0)) to clean the column, and then elute the bound antibody with elution buffer (0.1M glycine-HCl (pH 2.7)). Immediately neutralize the eluent with neutralization buffer (1M Tris-HCl (pH 9.0)). Select the eluted fraction of the antibody by DC protein analysis (BIO-RAD). After gathering together, the eluted fraction is concentrated to about 2mL with Centriprep-YM10 (Millipore). Next, the concentrated solution is supplied to gel filtration using a Superdex200 26/60 (Pharmacia) balanced with a 20mM acetate buffer (pH 6.0) containing 150mM NaCl. Reclaim the peak of the monomer component of the eluent and concentrate this component with Centriprep-YM10. The concentrate was filtered through a MILLEX-GW 0.22 μm filter unit (Millipore) and stored at 4° C. The concentration of the purified antibody was determined by converting the absorbance measured at a wavelength of 280 nm into a molar absorptivity.

[Reference Example 24] Binding to humanized anti-glypican 3 antibody produced in FT-KO cells Analysis of sugar chains

(1) Preparation of 2-aminobenzamide-labeled sugar chains (2-AB-labeled sugar chains)

By allowing N-glycosidase F (Roche diagnostics) to react with antibodies produced by FT-KO cells of the present invention and antibodies produced by CHO cells as a control sample, the sugar chains bound to the antibodies were released from the protein (Weitzhandler M. et al., Journal of Pharmaceutical Sciences (1994) 83(12), 1670-5). After removing the protein with ethanol (Schenk B. et al., The Journal of Clinical Investigation (2001), 108(11), 1687-95), the free sugar chains were concentrated to dryness and then fluorescently labeled with 2-aminopyridine (Bigge J.C. et al., Analytical Biochemistry (1995) 230(2), 229-238). The resulting 2-AB-linked sugar chains were de-reacted by solid-phase extraction using a cellulose column and then concentrated by centrifugation to be used for subsequent analysis in the form of purified 2-AB-linked sugar chains. Next, the purified 2-AB-modified sugar chains were allowed to react with β-galactosidase (Seikagaku Kogyo) to prepare non-galactosyl 2-AB-modified sugar chains.

(2) Analysis of non- galactosyl 2-AB sugar chains using normal phase HPLC

According to the method described above, free sugar chains from the FT-KO cell-produced antibody of the present invention and the CHO cell-produced antibody as a control sample were used as starting materials to prepare non-galactosyl 2-AB-linked sugar chains. These sugar chains were analyzed by normal phase HPLC using an amide column, TSKgel Amide-80 (Tosoh Co.), and the chromatograms were compared. In the CHO cell-produced antibody, G(0) was present as the main component of its sugar chain, and calculations based on the peak area ratio showed that about 4% of the total sugar chain contained G(0)-Fuc, which did not contain fucose. In contrast, in the FT-KO cell-produced antibody, G(0)-Fuc was the main component, and calculations based on the peak area ratio showed that more than 90% of the total sugar chain contained fucose-free sugar chains in the antibody produced by either strain.

[Table 17]

Relative ratio of each sugar chain in the non-galactosyl 2-AB sugar chain estimated by normal phase HPLC analysis

[Example 25] Establishment of Stable Expression Strains of Humanized H0L0 Antibody and Its Point Mutation Modified Antibodies

Genes encoding the Hspu2.2Lspu2.2 (Hu2.2Lu2.2) and Hspd1.8Lspd1.6 (Hd1.8Ld1.6) antibodies, or genes encoding the modified H0L0 antibodies, prepared according to the method described in Example 21, were cloned into expression vectors. During cloning, the genes encoding the H and L chains were inserted into separate expression vectors, allowing expression of the genes encoding the H and L chains that constitute the antibody. As described above, two expression vectors selected to form the desired combination of genes encoding the H and L chains were digested with PvuI and then introduced into the FTP-KO strain prepared in Reference Example 22 by electroporation.

When a transduced strain for stably producing H0L0 antibodies and modified antibodies thereof is prepared by electroporation, GenePulserII (Bio Rad) is used. 10 μg of each H chain and L chain expression plasmid DNA is mixed with 0.75 mL of CHO cells (1×10 ⁷ cells/mL) suspended in PBS to form the desired combination of H chain and L chain, and the mixture is allowed to stand on ice for 10 minutes. The mixture is moved to a Gene PulserII cup, and then an electric pulse is given with a capacity of 1.5 kV and 25 μFD. The mixture to which the electric pulse is given is allowed to stand at room temperature for 10 minutes, and then suspended in CHO-S-SFMII/1% HT/1% PS culture medium. 100 μL of 5, 10, and 50 times diluted suspensions prepared with the same culture medium are injected into each well of a 96-well culture plate. The plate is cultured in a CO ₂ incubator maintaining a 5% CO ₂ concentration for 24 hours. Afterwards, in each hole, add Geneticin (GIBCO) and Zeocin (Invitrogen), make final concentration reach 500 μ g/mL and 600 μ g/mL respectively, this plate is cultivated for 2 weeks again.The transduced cell colonies that will demonstrate Geneticin and Zeocin (bleomycin) resistance are gone down to posterity with the same culture medium containing 500 μ g/mL Geneticin (GIBCO) and 600 μ g/mL Zeocin (Invitrogen), thereby further screening.By using BiacoreQ (BIACORE) to evaluate the antibody concentration in this transduced cell culture supernatant screened as above, set up the transduced strain of highly expressed desired antibody.The mensuration of antibody concentration in the culture supernatant is carried out according to the accompanying operating instructions in BiacoreQ (BIACORE).

[Example 26] Efficacy testing of humanized H0L0 antibody and its point mutation-modified antibodies using an in vivo model

(1) Maintenance of cell lines transplanted into in vivo models

Hep G2 cells (ATCC) were used and maintained by passage in Minimun Essential Medium Eagle medium (SIGMA) containing 10% FBS, 1 mmol/l MEM sodium pyruvate (Invitrogen), and 1 mmol/l MEM non-essential amino acids (Invitrogen) (hereinafter referred to as passage medium).

(2) Preparation of Hep G2 cell transplantation mouse model

A cell suspension of Hep G2 cells was prepared using a solution containing a culture medium and MATRIGEL Matrix (BD Bioscience) (1:1) to a concentration of 5 × 10 ⁷ cells/mL. 100 μL of this cell suspension (5 × 10 ⁶ cells/mouse) was transplanted subcutaneously into the abdomen of SCID mice (male, 5 weeks old) (CLEA, Japan). The SCID mice were intraperitoneally administered 100 μL of anti-asialo GM1 antibody (Wako Pure Chemical Industries, Ltd., the contents of one vial were dissolved in 5 mL of this solution) one day before cell transplantation. Tumor volume was calculated using the formula: tumor volume = major diameter × minor diameter × minor diameter/2. The model was considered established when the average tumor volume was 130-330 mm ³ .

(3) Preparation of administration samples containing each test antibody

Dosing samples including the H0L0 antibody, Hu2.2Lu2.2 antibody, and Hd1.8Ld1.6 antibody were prepared with physiological saline at 0.5 mg/mL (5 mg/kg dosing group) or 0.1 mg/mL (1 mg/kg dosing group) on the day of administration.

(4) Administration of a sample containing an antibody

After transplanting Hep G2 cells into the mouse model prepared in (2), the administration sample prepared in (3) above was administered into the tail vein at a dosage of 10 mL/kg once a week for 3 weeks starting from the 27th day. As a negative control, physiological saline was also administered into the tail vein at a dosage of 10 mL/kg once a week for 3 weeks. All groups consisted of 5 mice, and each group was given an administration sample containing each antibody to be tested. Almost at the same time as the administration, venous blood was collected from 3 individuals in each group as a test substance for measuring the concentration of each antibody in the mouse blood. Specifically, blood was collected from the dorsal mid-foot vein at two time points: 0.5 hours after the initial administration and just before the second administration. The 20 μL volume of blood collected was treated with heparin and plasma was prepared by centrifugation.

(5) Evaluation of the anti-tumor effect of each tested antibody

The antitumor effects of each test antibody in a human liver cancer transplant mouse model were evaluated by measuring tumor volume one week after the last day of sample administration. As shown in Figure 55 , the Hspd1.8Lspd1.6 (Hd1.8Ld1.6) antibody showed a trend toward enhanced efficacy, while the Hspu2.2Lspu2.2 (Hu2.2Lu2.2) antibody showed a trend toward diminished efficacy.

(6) Concentration of each tested antibody in blood

The concentration of the test antibody in mouse plasma was determined using the ELISA method described in Example 21. Standard curve samples were prepared with plasma concentrations of 12.8, 6.4, 3.2, 1.6, 0.8, 0.4, and 0.2 μg/mL. The standard curve samples and mouse plasma test samples, appropriately diluted to the desired concentrations, were injected into a microplate (Nunc-Immuno Plate, MaxiSoup (Nalge nunc International)) immobilized with soluble Glypican-3 core (manufactured by Chugai Pharmaceutical Co., Ltd.). The plate was allowed to stand at room temperature for 1 hour. Then, goat anti-human IgG-BIOT (Southern Biotechnology Associates) and streptavidin-alkaline phosphatase conjugate (Roche Diagnostics) were injected sequentially, and a colorimetric reaction was performed using the BluePhos Microwell Phosphatase Substrate System (Kirkegaard & Perry Laboratories) as a substrate. The color development of the reaction solution in each well was calculated by measuring the absorbance of the reaction solution at 650 nm using a microplate reader. The antibody concentration in mouse plasma was calculated using the analysis software SOFTmax PRO (Molecular Devices) based on the calibration curve created from the absorbance of each calibration curve sample.

The mouse plasma concentrations 30 minutes and 7 days after administration are shown in Figure 56. The results showed that at all doses of the test antibody, as the pI of the test antibody further decreased, the mouse plasma antibody concentration increased 7 days after administration.

[Example 27] ADCC activity of each test antibody using human peripheral blood mononuclear cells as effector cells

Using human peripheral blood mononuclear cells (hereinafter referred to as human PBMC) as effector cells, the ADCC activity of each test antibody was measured as follows.

(1) Preparation of human PBMC solution

50 mL of peripheral blood was collected from a healthy volunteer (adult male) from Chugai Pharmaceutical Co., Ltd. using a syringe prefilled with 200 μL of a 1000 unit/mL heparin solution (Novo-Heparin Injection 5000 Units, Novo Nordisk). Four equal portions of this peripheral blood, diluted 2-fold with PBS(-), were added to Leucosep lymphocyte separation tubes (Greinerbio-one) that had been prefilled with 15 mL of Ficoll-Paque PLUS and centrifuged. The tubes containing peripheral blood were centrifuged at 2150 rpm for 10 minutes at room temperature to separate and collect the mononuclear cell fraction. The cells contained in each fraction were washed once with Dulbecco's Modified Eagle's Medium (SIGMA) supplemented with 10% FBS (hereinafter referred to as 10% FBS/D-MEM) and then suspended in 10% FBS/D-MEM to a cell density of 5 × 10 ⁶ /mL. This cell suspension was used as a human PBMC solution for subsequent experiments.

(2) Preparation of target cells

Hep G2 cells were detached from culture dishes and seeded into 96-well U-bottom plates at a density of 1× ^10⁴ cells/well. The plates were incubated overnight at 37°C in a 5% _CO₂ incubator. The next day, 5.55 MBq of Cr-51 was added to each well of the plate, and the plates were incubated at 37°C in a 5% _CO₂ incubator for 3 hours. The Hep G2 cells present in each well of the plate served as target cells for subsequent ADCC activity measurements.

(3) Chromium free test (ADCC activity)

ADCC activity was evaluated by the specific chromium release rate in the chromium release method. The target cells prepared in (2) were washed with culture medium, and 100 μL of H0L0 antibody, Hu2.2Lu2.2 antibody, and Hd1.8Ld1.6 antibody prepared at various concentrations (0, 0.004, 0.04, 0.4, 4, and 40 μg/mL) were added. The plate was reacted at room temperature for 15 minutes, and then the antibody solution was removed. Next, the plate with 100 μL of culture medium added to each well was incubated in a 5% CO ₂ incubator at 37°C for 1 hour. The plate with 100 μL of the human PBMC solution prepared in (1) added to each well (5×10 ⁵ cells/well) was allowed to stand in a 5% CO ₂ incubator at 37°C for 4 hours, and then centrifuged. The radioactivity of 100 μL of the culture supernatant in each well of the plate was measured using a γ counter. The specific chromium release rate was calculated according to the following formula: specific chromium release rate (%) = (AC) × 100/(BC).

In the above formula, A represents the average radioactivity (cpm) of 100 μL of culture supernatant in each well. B represents the average radioactivity (cpm) of 100 μL of culture supernatant in wells containing 100 μL of 2% NP-40 aqueous solution (Nonidet P-40, Nacalai Tesque) and 50 μL of 10% FBS/D-MEM medium added to the target cells. C represents the average radioactivity (cpm) of 100 μL of culture supernatant in wells containing 150 μL of 10% FBS/D-MEM medium added to the target cells. The assay was performed in triplicate, and the average and standard deviation of the specific chromium release rate (%) in the above assay, reflecting the ADCC activity of each test antibody, were calculated.

(4) Evaluation of ADCC activity of each test antibody

The ADCC activity of human PBMCs exerted by each of the tested antibodies was evaluated, and as a result, it was confirmed that all of the tested antibodies had ADCC activity. The results are shown in Figure 57. A significance test was performed on the specific chromium free rate displayed by each of the tested antibodies at each concentration, and the results confirmed that there was no significant difference in the specific chromium free rate displayed by each of the tested antibodies at all antibody concentrations. The SAS preclinical package (SAS Institute Inc.) was used in the statistical analysis. The above results show that there was no difference in the ADCC activity of each of the tested antibodies with changed pI.

[Example 28] Preparation of anti-human IL-6 receptor antibodies, anti-human GPC3 antibodies, and anti-human IL-31 receptor antibodies

1. Preparation of anti-human IL-6 receptor antibody

Two anti-human IL-6 receptor antibodies were prepared. 6R_a_H1L1, composed of 6R_a_H1 (SEQ ID NO: 221) as the H chain and 6R_a_L1 (SEQ ID NO: 224) as the L chain, and 6R_b_H1L1, composed of 6R_b_H1 (SEQ ID NO: 227) as the H chain and 6R_b_L1 (SEQ ID NO: 229) as the L chain, were prepared. Animal cell expression vectors encoding the respective amino acid sequences were prepared according to Reference Examples 1 and 2, and the antibodies were expressed and purified.

2. Preparation of anti-human GPC3 antibody

Anti-human GPC3 antibodies were prepared. GPC3_H1L1, composed of GPC3_H1 (SEQ ID NO: 233) as the H chain and GPC3_L1 (SEQ ID NO: 236) as the L chain, was prepared. Animal cell expression vectors encoding the respective amino acid sequences were prepared according to Reference Examples 1 and 2, and the antibodies were expressed and purified.

3 anti-human IL-31 receptor antibody

Anti-human IL-31 receptor antibodies were prepared. 31R_H1L1, composed of 31R_H1 (SEQ ID NO: 239) as the H chain and 31R_L1 (SEQ ID NO: 242) as the L chain, was prepared. Animal cell expression vectors encoding the respective amino acid sequences were prepared according to Reference Examples 1 and 2, and the antibodies were expressed and purified.

[Example 29] Reduction of the expression of anti-human IL-6 receptor antibodies, anti-human GPC3 antibodies, and anti-human IL-31 by amino acid substitution Isoelectric point of receptor antibody

1. Explore CDR sequences that lower the isoelectric point without reducing antigen binding activity

WO/2007/114319 provides an example of using amino acid substitutions in CDRs to control the isoelectric point. Amino acid substitutions were performed in the H chain CDR3. However, since the H chain CDR3 is closely related to the antibody's antigen-binding activity, it was assumed that amino acid substitutions at the same position would neither diminish antigen-binding activity nor lower the isoelectric point, depending on the antibody species. Therefore, a search was conducted for candidate CDR sequences that could lower the isoelectric point without diminishing antigen-binding activity, regardless of the antibody species. The results identified candidate CDR sequences that could lower the isoelectric point without diminishing antigen-binding activity as candidate CDR sequences in the H chain variable region: H31, H52, H61, H62, H64, and H65; and L24, L27, L27a, L53, L54, L55, and L56 (Kabat numbering) in the L chain variable region. Therefore, amino acid substitutions were made in several of the candidate CDR sequences in the anti-human IL-6 receptor antibody, anti-human GPC3 antibody, and anti-human IL-31 receptor antibody shown below to investigate whether the isoelectric point could be lowered without impairing antigen-binding activity.

2. Preparation of Anti-Human IL-6 Receptor Antibodies with Lowered Isoelectric Points, Binding Activity Evaluation, and Isoelectric Point Determination

Amino acid substitutions that lower the isoelectric point and other amino acid substitutions were introduced into 6R_a_H1 (SEQ ID NO: 221) and 6R_a_L1 (SEQ ID NO: 224), respectively, constituting the anti-human IL-6 receptor antibody 6R_a_H1L1, to construct 6R_a_H2 (SEQ ID NO: 222) and 6R_a_L2 (SEQ ID NO: 225). Vectors were prepared according to the methods of Reference Examples 1 and 2, and 6R_a_H2L2 was expressed and purified. Amino acid substitutions that lower the isoelectric point and other amino acid substitutions were further introduced into 6R_a_H2L2 to construct 6R_a_H3 (SEQ ID NO: 223) and 6R_a_L3 (SEQ ID NO: 226). Vectors were prepared according to the method of Reference Example 1, and 6R_a_H3L3 was expressed and purified.

The dissociation constants (KD) of 6R_a_H1L1, 6R_a_H2L2, and 6R_a_H3L3 for the human IL-6 receptor as an antigen were measured using the Biacore T100 method described in Reference Example 3. As shown in Table 18 below, the dissociation constants (KD) of 6R_a_H1L1, 6R_a_H2L2, and 6R_a_H3L3 for the IL-6 receptor were comparable, and no significant reduction in antigen-binding activity was observed with the introduction of amino acid substitutions.

[Table 18]

The isoelectric points of 6R_a_H1L1 were determined using isoelectric electrophoresis, a method known to those skilled in the art. The results were as follows: The isoelectric point of 6R_a_H2L2, which had amino acid substitutions that lowered the isoelectric point, was approximately 6.1, and the isoelectric point of 6R_a_H3L3 was approximately 5.4, representing decreases of approximately 3.1 and 3.8, respectively, compared to 6R_a_H1L1. Furthermore, the theoretical isoelectric points of the variable regions VH/VL were calculated using GENETYX (GENETYX CORPORATION). The theoretical isoelectric point of 6R_a_H1L1 was 9.37, while that of 6R_a_H2L2 was 4.63, and that of 6R_a_H3L3 was approximately 4.27, representing decreases of 4.74 and 5.10, respectively, compared to 6R_a_H1L1. The above results are summarized in Table 19.

[Table 19]

The amino acid substitutions introduced into the CDR sequence of 6R_a_H1L1 are summarized in the following Table 20. The results show that these CDR amino acid substitutions can lower the isoelectric point of the anti-human IL-6 receptor antibody 6R_a_H1L1 without significantly reducing the antigen-binding activity.

[Table 20]

Subsequently, amino acid substitutions that lower the isoelectric point and other amino acid substitutions were introduced into 6R_b_H1 (SEQ ID NO: 227) and 6R_b_L1 (SEQ ID NO: 229), constituting another anti-human IL-6 receptor antibody 6R_b_H1L1, to construct 6R_b_H2 (SEQ ID NO: 228) and 6R_b_L2 (SEQ ID NO: 230). Vectors were prepared according to the methods of Reference Examples 1 and 2, and 6R_b_H2L2 was expressed and purified. Furthermore, amino acid substitutions that lower the isoelectric point and other amino acid substitutions were introduced into 6R_b_H2L2 to construct 6R_b_L3 (SEQ ID NO: 231) and 6R_b_L4 (SEQ ID NO: 232). Vectors were prepared according to the method of Reference Example 1, and 6R_b_H2L3 and 6R_b_H2L4 were expressed and purified.

The neutralizing activity of 6R_b_H1L1, 6R_b_H2L2, 6R_b_H2L3, and 6R_b_H2L4 against the human IL-6 receptor as an antigen was measured according to the method described in Reference Example 4. As shown in FIG58 , the neutralizing activities of 6R_b_H1L1, 6R_b_H2L2, 6R_b_H2L3, and 6R_b_H2L4 were almost equivalent, and no significant decrease in antigen-binding activity was observed due to the introduction of amino acid substitutions.

The isoelectric point was determined using isoelectric electrophoresis, a method known to those skilled in the art. The results were as follows: the isoelectric point of 6R_b_H1L1 was approximately 9.3. In contrast, the isoelectric point of 6R_b_H2L2, which had undergone amino acid substitutions to lower the isoelectric point, was approximately 5.9. Compared with 6R_b_H1L1, the isoelectric point of 6R_b_H2L2 was reduced by approximately 3.4. Furthermore, when the theoretical isoelectric points of the variable regions VH/VL were calculated using GENETYX (GENETYX CORPORATION), the theoretical isoelectric point of 6R_b_H1L1 was 9.20. In contrast, the theoretical isoelectric points of 6R_b_H2L2 were 4.52, 6R_b_H2L3 were approximately 4.46, and 6R_b_H2L4 were approximately 4.37. Compared to 6R_b_H1L1, the theoretical isoelectric points of the three were lower by 4.68, 4.74, and 4.83, respectively. These results are summarized in Table 21.

[Table 21]

N.T.: Not quantifiable

The amino acid substitutions introduced into the CDR sequence of 6R_b_H1L1 are summarized in the following Table 22. For the anti-human IL-6 receptor antibody 6R_b_H1L1, it was found that these CDR amino acid substitutions could lower the isoelectric point of the antibody molecule without significantly reducing the antigen-binding activity.

[Table 22]

3. Preparation of Anti-Human GPC3 Antibodies with Lowered Isoelectric Points, Binding Activity Evaluation, and Isoelectric Point Determination

Amino acid substitutions that lower the isoelectric point and other amino acid substitutions were introduced into GPC3_H1 (SEQ ID NO: 233) and GPC3_L1 (SEQ ID NO: 236), respectively, constituting the anti-human GPC3 antibody GPC3_H1L1, to construct GPC3_H2 (SEQ ID NO: 234) and GPC3_L2 (SEQ ID NO: 237). Vectors were prepared according to the methods of Reference Examples 1 and 2, and GPC3_H2L2 was expressed and purified. Amino acid substitutions that lower the isoelectric point and other amino acid substitutions were further introduced into GPC3_H2L2 to construct GPC3_H3 (SEQ ID NO: 235) and GPC3_L3 (SEQ ID NO: 238). Vectors were prepared according to the method of Reference Example 1, and GPC3_H3L3 was expressed and purified.

The binding activity of GPC3-H1L1, GPC3-H2L2, and GPC3-H3L3 to human GPC3 as an antigen was evaluated using the competitive ELISA method described in Reference Example 5. The results are shown in Figures 59 and 60. The binding activity of GPC3-H1L1, GPC3-H2L2, GPC3-H2L2, and GPC3-H3L3 to glypican 3 was almost identical, and no significant decrease in antigen-binding activity was observed due to the introduction of amino acid substitutions.

The isoelectric point was determined using isoelectric electrophoresis, a method known to those skilled in the art. The results were as follows: GPC3_H1L1 had an isoelectric point of approximately 9.6, while GPC3_H2L2, which had undergone amino acid substitutions that lowered the isoelectric point, had an isoelectric point of approximately 8.9, representing a decrease of 0.7 points compared to the isoelectric point of GPC3_H1L1. Similarly, GPC3_H2L2 had an isoelectric point of approximately 8.7, while GPC3_H3L3, which had undergone amino acid substitutions that lowered the isoelectric point, had an isoelectric point of approximately 6.5, representing a decrease of 2.2 points compared to the isoelectric point of GPC3_H2L2. Furthermore, the theoretical isoelectric point of GPC3_H1L1 was 9.65, while that of GPC3_H2L2 was 8.47, a decrease of 1.18 points compared to GPC3_H1L1. Similarly, the theoretical isoelectric point of GPC3_H2L2 was 8.47, while that of GPC3_H3L3 was 4.93, a decrease of 3.54 points compared to GPC3_H2L2. These results are summarized in Table 23.

[Table 23]

The amino acid substitutions introduced into the CDR sequence of GPC3_H1L1 are summarized in the following Table 24. It was found that these CDR amino acid substitutions can lower the isoelectric point of the anti-human GPC3 antibody GPC3_H1L1 without significantly reducing the antigen-binding activity.

[Table 24]

4 Anti-human IL-31 receptor antibodies with lowered isoelectric points, binding activity evaluation, and isoelectric point determination

Amino acid substitutions that lower the isoelectric point and other amino acid substitutions were introduced into 31R_H1 (SEQ ID NO: 239) and 31R_L1 (SEQ ID NO: 242), respectively, constituting 31R_H1L1, to construct 31R_H2 (SEQ ID NO: 240) and 31R_L2 (SEQ ID NO: 243). Vectors were prepared according to the methods of Reference Examples 1 and 2, and 31R_H2L2 was expressed and purified. Amino acid substitutions that lower the isoelectric point and other amino acid substitutions were further introduced into 31R_H2L2 to construct 31R_H3 (SEQ ID NO: 241). Vectors were prepared according to the method of Reference Example 1, and 31R_H3L2 was expressed and purified.

The binding activity of 31R_H2L2 and 31R_H3L2 to IL-31 was evaluated using the Biacore method described in Reference Example 6, and the results are summarized in Table 25. As shown in Table 25, the binding activity of 31R_H2L2 and 31R_H3L2 was almost equivalent to that of NR10 of 31R_H1L1, and no significant decrease in antigen-binding activity was observed due to the introduction of amino acid substitutions.

[Table 25]

The isoelectric point was determined by isoelectric electrophoresis using a method known to those skilled in the art. The results were as follows: the isoelectric point of 31R_H1L1 was approximately 7.76. In contrast, the isoelectric point of 31R_H2L2, which had undergone amino acid substitutions to lower the isoelectric point, was approximately 5.49, and the isoelectric point of 31R_H3L2 was approximately 5.43, which were approximately 2.27 and 2.33 lower than those of 31R_H1L1, respectively. Furthermore, the theoretical isoelectric point of 31R_H1L1 was 7.76, while the theoretical isoelectric point of 31R_H2L2 was 4.63, and the theoretical isoelectric point of 31R_H3L2 was approximately 4.54, which were approximately 3.13 and 3.22 lower than those of 31R_H1L1, respectively. These results are summarized in Table 26.

[Table 26]

The amino acid substitutions introduced into the CDR sequence of 31R_H1L1 are summarized in the following Table 27. For the anti-human IL-31 receptor antibody 31R_H1L1, it was found that these CDR amino acid substitutions could lower the isoelectric point of the antibody molecule without significantly reducing the antigen-binding activity.

[Table 27]

5 can reduce the anti-human IL-6 receptor antibody, anti-human GPC3 antibody, anti- CDR sequences of human IL-31 receptor antibodies with isoelectric points

The H chain CDR sequences of the two anti-human IL-6 receptor antibodies (6R_a and 6R_b), anti-human GPC3 antibody (GPC3), and anti-human IL-31 receptor antibody (31R) produced in the above studies are summarized in Table 28, and the L chain CDR sequences are summarized in Table 29. Amino acid substitutions that can lower the isoelectric point without impairing antigen-binding activity are indicated by shading.

[Table 28]

[Table 29]

This revealed that H31, H61, H62, H64, and H65 in the H chain variable region and L24, L27, L53, L54, and L55 (Kabat numbering) in the L chain variable region are common CDR sites in multiple antibodies at which amino acid substitutions that lower the antibody isoelectric point can be introduced without significantly reducing the antibody's antigen-binding activity, regardless of the antibody species.

WO/2007/114319 demonstrates that lowering the isoelectric point of an antibody can improve the pharmacokinetics of IgG. In WO/2007/114319, amino acid substitutions were primarily made within the framework of the antibody variable region, without diminishing antigen-binding activity. The measured isoelectric point of the anti-Factor IXa antibody shifted by approximately 0.9, while the theoretical isoelectric point shifted by approximately 1.0. The measured isoelectric point of the anti-Factor X antibody shifted by approximately 0.5, while the theoretical isoelectric point shifted by approximately 0.1, indicating minimal shifts in isoelectric point.

The present invention discovered CDR sequences that do not diminish antigen-binding activity, allowing amino acid substitutions to be introduced not only into the framework of the antibody variable region but also into the CDRs to lower the isoelectric point. This discovery resulted in a decrease in the measured isoelectric point of the anti-human IL-6 receptor antibody by approximately 3.8 and a theoretical isoelectric point of approximately 5.1; in the anti-human GPC3 antibody, a decrease in the measured isoelectric point by approximately 3.1 and a theoretical isoelectric point of approximately 4.7; and in the anti-human IL-31 receptor antibody, a decrease in the measured isoelectric point by approximately 3.2 and a theoretical isoelectric point by approximately 2.3, significantly lowering the isoelectric point compared to amino acid substitutions made only within the framework.

[Example 30] Evaluation of anti-human IL-6 receptor antibodies, anti-human GPC3 antibodies, and anti-human IL-31 receptor antibodies with lowered isoelectric points Antibody Pharmacokinetics

1 Evaluation of the pharmacokinetics of anti-human IL-6 receptor antibodies in cynomolgus monkeys and mice

The pharmacokinetics of the anti-human IL-6 receptor antibody 6R_a_H1L1 and the isoelectrically lowered anti-human IL-6 receptor antibodies 6R_a_H2L2 and 6R_a_H3L3 were evaluated in cynomolgus monkeys. Both 6R_a_H1L1 and 6R_a_H2L2 were administered intravenously as a single dose at 1.0 mg/kg, and blood samples were collected before and after administration. Additionally, both 6R_a_H2L2 and 6R_a_H3L3 were administered subcutaneously as a single dose at 1.0 mg/kg, and blood samples were collected before and after administration.

Plasma concentration determination is carried out according to the ELISA method. The standard curve sample and plasma determination sample of appropriate concentration are injected into a microplate (Nunc-ImmunoPlate, MaxiSorp (Nalge nunc International)) fixed with anti-human IgG (γ chain specific) F (ab') 2 (Sigma), and after standing at room temperature for 1 hour, goat anti-human IgG-BIOT (Southern Biotechnology Associates) and streptavidin alkaline phosphatase conjugate (Roche Diagnostics) are reacted with it in sequence, and a color reaction is carried out using the BluePhos Microwell phosphatase substrate system (Kirkegaard & Perry Laboratories) as a substrate, and the absorbance at 650 nm is measured with a microplate reader. Using analysis software SOFTmax PRO (Molecular Devices), the plasma concentration is calculated by the absorbance of the standard curve. The obtained plasma concentration change data were subjected to model-independent analysis using the pharmacokinetic analysis software WinNonlin (manufactured by Pharsight), and the clearance (CL) was calculated. The results are shown in Table 30. Comparison of intravenously administered 6R_a_H1L1 and 6R_a_H2L2 confirmed that the clearance of 6R_a_H2L2, whose isoelectric point was lowered, was lower, and that its pharmacokinetics were improved by lowering the isoelectric point. Comparison of subcutaneously administered 6R_a_H2L2 and 6R_a_H3L3 confirmed that the clearance of 6R_a_H3L3, whose isoelectric point was lowered, was lower, and that its pharmacokinetics were improved by lowering the isoelectric point.

[Table 30]

Next, the pharmacokinetics of different anti-human IL-6 receptor antibodies, 6R_b_H1L1, and 6R_b_H2L2, an anti-human IL-6 receptor antibody with a lowered isoelectric point, were evaluated in mice (C57BL/6J, Charles River, Japan). Both 6R_b_H1L1 and 6R_b_H2L2 were administered intravenously as a single dose at 1.0 mg/kg, and blood samples were collected before and after administration. Additionally, both 6R_b_H1L1 and 6R_b_H2L2 were administered subcutaneously as a single dose at 1.0 mg/kg, and blood samples were collected before and after administration.

The concentration in plasma was determined by ELISA. First, recombinant human IL-6 sR (R&D Systems) was biotinylated using the EZ-LinkTM Sulfo-NFS-biotinylation kit (PIERCE). The biotinylated human-sIL-6R was injected into a plate coated with Reacti-Bind high binding energy streptavidin (HBC) (PIERCE), and allowed to stand for more than 1 hour at room temperature to form a human-sIL-6R solidification plate. Standard curve samples and mouse plasma assay samples of appropriate concentrations were prepared, injected into human-sIL-6R solidification plates, and allowed to stand for 1 hour at room temperature. Afterwards, anti-human IgG-AP (SIGMA) was reacted with it, and a color reaction was performed using the BluePhos Microwell phosphatase substrate system (Kirkegaard & Perry Laboratories) as a substrate, and the absorbance at 650 nm was measured using a microplate reader. Plasma concentrations were calculated from the absorbance of the standard curve using the analysis software SOFTmax PRO (Molecular Devices). The resulting plasma concentration change data were subjected to model-independent analysis using the pharmacokinetic analysis software WinNonlin (Pharsight) to calculate clearance (CL). The results are shown in Table 31. Comparison of intravenous and subcutaneous administration of 6R_a_H1L1 and 6R_a_H2L2 confirmed that the clearance of 6R_a_H2L2, whose isoelectric point was lowered, was lower in both cases, indicating that lowering the isoelectric point improves its pharmacokinetics.

[Table 31]

2 Evaluation of the pharmacokinetics of anti-human GPC3 antibodies in mice

The pharmacokinetics of the anti-human GPC3 antibody GPC3_H1L1 and the isoelectrically lowered anti-human GPC3 antibodies GPC3_H2L2 and GPC3_H3L3 were evaluated in C.B-17/Icr-scid mice. GPC3_H1L1, GPC3_H2L2, and GPC3_H3L3 were each administered a single intravenous dose of 5.0 mg/kg, and blood was collected before and after administration.

The concentration in plasma was determined by ELISA. The standard curve sample of the appropriate concentration and the mouse plasma test sample appropriately diluted to the desired concentration were injected into a microplate (Nunc-Immuno Plate, MaxiSoup (Nalge nunc International)) fixed with the antigen GPC3 (Chugai Pharmaceutical Co., Ltd.), and the plate was left to stand at room temperature for 1 hour. Afterwards, goat anti-human IgG-BIOT (Southern Biotechnology Associates) and streptavidin alkaline phosphatase conjugate (Roche Diagnostics) were injected in sequence, and a color reaction was performed using the BluePhos Microwell phosphatase substrate system (Kirkegaard & Perry Laboratories Co., Ltd.) as a substrate, and the absorbance at 650 nm was measured with a microplate reader. The concentration in plasma was calculated by the absorbance of the standard curve using analysis software SOFTmax PRO (Molecular Devices Co., Ltd.). The obtained plasma concentration change data were subjected to model-independent analysis using the pharmacokinetic analysis software WinNonlin (manufactured by Pharsight), and the clearance (CL) was calculated. The results are shown in Table 32. When GPC3_H1L1 and GPC3_H2L2 were compared, the clearance of GPC3_H2L2, whose isoelectric point was lowered, was lower. When GPC3_H2L2 and GPC3_H3L3 were compared, the clearance of GPC3_H3L3, whose isoelectric point was further lowered, was lower, indicating that the pharmacokinetics were improved by lowering the isoelectric point.

[Table 32]

3 Evaluation of the pharmacokinetics of anti-human IL-31 receptor antibodies in mice

The pharmacokinetics of the anti-human IL-31 receptor antibody 31R_H1L1 and the isoelectrically lowered anti-human IL-31 receptor antibody 31R_H2L2 were evaluated in mice (C57BL/6J, Charles River, Japan). Both 31R_H1L1 and 31R_H2L2 were administered intravenously as a single dose of 1.0 mg/kg, and blood was collected before and after administration.

Plasma concentration determination is carried out by ELISA method. The standard curve sample and plasma determination sample of appropriate concentration are injected into the microplate (Nunc-Immuno Plate, MaxiSorp (Nalge nunc International Society)) solidified with anti-human IgG (Fc specificity) antibody (Sigma Society system) respectively, after standing at room temperature for 1 hour, goat anti-human IgG-ALP (Sigma Society system) is reacted with it at room temperature for 1 hour, afterwards using BluePhos Microwell phosphatase substrate system (Kirkegaard＆Perry Laboratories Society system) as substrate to carry out color reaction, measure the absorbance of 650nm with microplate reader. Use analysis software SOFTmax PRO (Molecular Devices Society system), by the absorbance of standard curve, calculate plasma concentration.

The obtained plasma concentration change data were subjected to model-independent analysis using the pharmacokinetic analysis software WinNonlin (manufactured by Pharsight) to calculate the clearance (CL). The results are shown in Table 33. Comparison of 31R_H1L1 and 31R_H2L2 confirmed that the clearance of 31R_H2L2, whose isoelectric point was lowered, was lower, and that the elimination rate was reduced by lowering the isoelectric point.

[Table 33]

3 Conclusion

In the present invention, the present inventors have found that: in multiple antibodies with different antigenic species, amino acid substitutions in CDR sequences can be used to reduce the isoelectric point of the antibody and improve the pharmacokinetics of the antibody without weakening the antigen-binding activity of the antibody. Among the amino acid substitutions in the CDR sequences found, H31, H61, H62, H64, H65 in the H chain variable region, L24, L27, L53, L54, L55 (Kabat numbering) in the L chain variable region do not depend on the antibody species. In multiple antibodies, amino acid substitutions that reduce the isoelectric point of the antibody without significantly weakening the antigen-binding activity of the antibody and amino acid substitutions in the CDR sequences that improve pharmacokinetics can be introduced. It is believed that these mutation sites in the CDR sequence can reduce the isoelectric point of the antibody without significantly weakening the antigen-binding activity of the antibody, and it is believed that they can be used as amino acid substitution sites that improve the pharmacokinetics of the antibody.

[Example 31] Anti-human IL-6 receptor antibody and anti-human GPC3 antibody were purified by standard chromatography to reduce the isoelectric point. Peak separation of heterodimers and homodimers of anti-human IL-31 receptor antibodies

1. Expression of anti-Factor IX antibody/anti-Factor X antibody heterodimers

Patent document WO/2007/114325 reports a method for purifying IgG-type bispecific antibodies with a common L chain. To express IgG-type bispecific antibodies with a common L chain, it is necessary to express the L chain that is common to both H chains (A chain and B chain). In this case, not only the A-chain and B-chain heterodimers of the target bispecific antibody are expressed, but also A-chain homodimers and B-chain homodimers. Therefore, the A-chain and B-chain heterodimers of the target bispecific antibody must be purified from a mixture of the three antibodies. The same patent states that, while conventional methods cannot separate the peaks of A-chain and B-chain heterodimers, A-chain homodimers, and B-chain homodimers using standard chromatography, and thus purify the A-chain and B-chain heterodimers, by substituting amino acids in the variable regions of the A and B chains, which are the two H chains, to introduce a difference in isoelectric point between the A-chain and B-chain homodimers, the peaks of A-chain and B-chain heterodimers, A-chain homodimers, and B-chain homodimers can be separated using cation exchange chromatography in standard chromatography, thereby purifying the A-chain and B-chain heterodimers. The same patent states that amino acid substitutions in the CDRs, which are variable region amino acids, are believed to affect the antigen-binding activity of the antibody, and therefore only framework amino acid substitutions are performed. However, as described above, framework amino acid substitutions cannot introduce a significant change in isoelectric point. Therefore, to efficiently purify the A-chain and B-chain heterodimers, it is preferable to introduce a larger difference in isoelectric point between the A-chain and B-chain homodimers. Therefore, it was examined whether the peaks of A chain and B chain heterodimers, A chain homodimers, and B chain homodimers could be separated by introducing the amino acid substitutions found in Example 28 that lower the antibody isoelectric point without significantly reducing the antibody's antigen-binding activity.

2. Expression of anti-human IL-6 receptor antibody/anti-human IL-6 receptor antibody with reduced pI heterodimer

Using 6R_a_H1 (SEQ ID NO: 221) as the A chain, 6R_a_H3 (SEQ ID NO: 223) in which the isoelectric point is lowered without reducing the antigen-binding activity and an isoelectric point difference with the A chain is introduced as the B chain, and 6R_a_L3 (SEQ ID NO: 226) as the common L chain, 6R_a_H1H3L3 (a mixture of A chain and B chain heterodimers (6R_a_H1/H3/L3), A chain homodimers (6R_a_H1/L3), and B chain homodimers (6R_a_H3/L3)) was expressed and purified according to the method described in Reference Example 2.

3. Expression of Heterodimers of Anti-Human GPC3 Antibodies/Anti-Human GPC3 Antibodies with Reduced pI

Using GPC3_H2 (SEQ ID NO: 234) as the A chain, GPC3_H3 (SEQ ID NO: 235) in which the isoelectric point was lowered without reducing the antigen-binding activity and a difference in isoelectric point from the A chain was introduced as the B chain, and GPC3_L3 (SEQ ID NO: 238) as the common L chain, GPC3_H2H3L3 (a mixture of A chain and B chain heterodimers (GPC3_H2/H3/L3), A chain homodimers (GPC3_H2/L3), and B chain homodimers (GPC3_H3/L3)) was expressed and purified according to the method described in Reference Example 2.

4. Expression of anti-human IL-31 receptor antibody/anti-human IL-31 receptor antibody with reduced pI heterodimer

Using 31R_H1a (SEQ ID NO: 244) in which the constant region of 31R_H1 was modified as the A chain, 31R_H2a (SEQ ID NO: 245) in which the constant region of 31R_H2 was similarly modified without reducing the antigen-binding activity and introducing a difference in isoelectric point from the A chain was used as the B chain, and 31R_L2 (SEQ ID NO: 243) was used as the common L chain. According to the method described in Reference Example 2, 31R_H1aH2aL2 (a mixture of A chain and B chain heterodimers (31R_H1a/H2a/L2), A chain homodimers (31R_H1a/L2), and B chain homodimers (31R_H2a/L2)) was expressed and purified.

5. Evaluation of expressed antibodies by cation exchange chromatography

The theoretical isoelectric point differences between the VH/VL of the A-chain homodimer and the B-chain homodimer of the antibodies prepared above are summarized in Table 34 below. By introducing amino acid substitutions not only into the H-chain framework but also into the CDR sequences that lower the isoelectric point while maintaining binding activity, a maximum difference of 1.56 between the theoretical isoelectric points of the A-chain homodimer and the B-chain homodimer can be introduced. Patent document WO/2007/114325 shows that by introducing amino acid substitutions only into the framework of the A-chain homodimer to lower the isoelectric point, and introducing amino acid substitutions only into the framework of the B-chain homodimer to increase the isoelectric point, a difference of 1.13 between the theoretical isoelectric points of the VH/VL of the A-chain homodimer and the B-chain homodimer can be introduced. This study demonstrates that, even when amino acid substitutions are made to only one chain (without lowering the isoelectric point), by introducing amino acid substitutions not only into the framework but also into the CDR sequences, a difference of up to 1.56 in the theoretical isoelectric point can be introduced. Specifically, by introducing amino acid substitutions not only into the framework but also into the CDR sequences for separating A-chain homodimers and B-chain homodimers while maintaining binding activity, the difference in isoelectric points between the two components can be further increased. Separation using standard ion exchange chromatography typically relies on the difference in isoelectric points between the two components to be separated, so this approach is believed to facilitate separation.

[Table 34]

The ability of peak separation of the A-chain and B-chain heterodimers, A-chain homodimers, and B-chain homodimers of 6R_a_H1H3L3, GPC3_H2H3L3, and 31R_H1aH2aL2 was evaluated by cation exchange chromatography. A ProPac WCX-10 (Dionex) standard cation exchange chromatography column was used, with 25mM MES (pH 5.0) as mobile phase A and 25mM MES, 1M NaCl (pH 5.0) as mobile phase B. Analysis was performed using an appropriate flow rate and gradient. The results of the cation exchange chromatography evaluation are shown in Figure 61. Peak separation of the A-chain and B-chain heterodimers, A-chain homodimers, and B-chain homodimers was observed in all of 6R_a_H1H3L3, GPC3_H2H3L3, and 31R_H1aH2aL2.

It was discovered that in the H chain variable region, H31, H61, H62, H64, and H65 (Kabat numbering) can lower the isoelectric point of the antibody without significantly reducing the antibody's antigen-binding activity, regardless of the antibody species, by amino acid substitution at the same site. This allows separation of heterodimers and homodimers by cation exchange chromatography. These mutation sites in the CDR sequence are believed to lower the isoelectric point of the antibody without significantly reducing the antibody's antigen-binding activity, regardless of the antigen species, and can be used as amino acid substitution sites to increase the difference in isoelectric point between heterodimers and homodimers of bispecific antibodies.

[Reference Example 1]

Preparation of antibody expression vector genes

The making of each mutant is carried out by assembling PCR using QuikChange site-directed mutagenesis kit (Stratagene) or PCR.When using QuikChange site-directed mutagenesis kit (Stratagene), mutant is made according to the method for appendix instructions.The method carried out using assembling PCR adopts any of the following methods to carry out.In the first method, the synthesis of oligoDNA designed according to the sequence of the positive chain and the reverse chain of the positive chain comprising the modification site is carried out.The positive chain oligoDNA comprising the modification site and the reverse chain oligoDNA combined with the vector inserted with the gene to be modified, the reverse chain oligoDNA comprising the modification site and the positive chain oligoDNA combined with the vector inserted with the gene to be modified are combined respectively, PCR is carried out using PrimeSTAR (TAKARA), thus making 5 end side and 3 end side fragments comprising the modification site.By assembling PCR, these 2 fragments are connected, thus making each mutant.In the second method, by making the oligoDNA of appropriate number to cover the whole variable region, assembling PCR is used afterwards that above-mentioned oligoDNA is connected, thus making the whole variable region. The mutant prepared according to the above method is inserted into an expression vector capable of expressing the inserted gene in animal cells, and the nucleotide sequence of the obtained expression vector is determined according to methods known to those skilled in the art.

[Reference Example 2]

Antibody expression and purification

The antibody was expressed using the following method. The HEK293H strain (Invitrogen) derived from human embryonic kidney cancer cells was suspended in DMEM medium (Invitrogen) containing 10% fetal bovine serum (Invitrogen). 10 mL of the suspension was seeded at a cell density of 5 to 6 × 10 ⁵ cells/mL in each culture dish for adherent cells (10 cm diameter, CORNING). The cells were cultured in an incubator at 37°C and 5% CO ₂ for one day and night. After that, the culture medium was aspirated and 6.9 mL of CHO-S-SFM-II (Invitrogen) medium was added. The prepared plasmid was introduced into the cells by lipid transfection. The obtained culture supernatant was recovered and then centrifuged at approximately 2000 g for 5 minutes at room temperature to remove the cells. The culture supernatant was then sterilized by passing through a 0.22 μm filter MILLEX(R)-GV (Millipore) to obtain the culture supernatant. Antibodies were purified from the resulting culture supernatant using rProtein A Sepharose™ Fast Flow (Amersham Biosciences) according to methods known to those skilled in the art. The concentration of the purified antibody was determined by measuring absorbance at 280 nm using a spectrophotometer. The absorbance coefficient was calculated from the obtained value using the PACE method, and the antibody concentration was calculated using this absorbance coefficient (Protein Science 1995; 4: 2411-2423).

[Reference Example 3]

Method for evaluating the affinity of anti-human IL-6 receptor antibodies to IL-6 receptor using Biacore

1. Preparation of Soluble Human IL-6 Receptor

Recombinant human IL-6 receptor, which serves as an antigen, was prepared as follows. A CHO cell constant expression strain of the soluble human IL-6 receptor consisting of the amino acid sequence from positions 1 to 344 on the N-terminal side reported in J. Biochem. 108, 673-676 (1990) (Yamasaki et al., Science 1988; 241: 825-828 (GenBank #X12830)) was prepared. The soluble human IL-6 receptor was purified from the culture supernatant obtained from CHO cells expressing the soluble human IL-6 receptor using three types of column chromatography: Blue Sepharose 6 FF column chromatography, affinity chromatography on a column immobilized with a soluble human IL-6 receptor-specific antibody, and gel filtration column chromatography. The fraction eluting as the main peak was the final pure product.

2. Evaluation of affinity for soluble human IL-6 receptor using Biacore

Using Biacore T100 (GE Healthcare Bioscience), the speed of the antigen-antibody reaction of anti-human IL-6 receptor antibodies and soluble human IL-6 receptor was analyzed. According to methods well known to those skilled in the art, rec-Protein A (ZYMED) (hereinafter referred to as protein A) was fixed on a sensor chip, the antibody was captured on the immobilized protein A, and then the antigen was reacted as an analyte to measure the interaction between the antibody and the antigen. The running buffer used was HBS-EP+, and the flow rate was 20 μL/min. Each antibody was prepared into about 100RU and bound to protein A/G. The soluble human IL-6 receptor used as the analyte was adjusted to 0, 0.065, 0.131, and 0.261 μg/mL with HBS-EP+. In the assay, the antibody solution was first bound to protein A/G, and the analyte solution was then allowed to interact with it for 3 minutes, after which it was switched to HBS-EP+ and the dissociation phase was measured for 10 or 15 minutes. After the dissociation phase, the sensor chip was washed with 10 μL of 10 mM glycine-HCl (pH 1.5) to regenerate the sensor chip. This binding, dissociation, and regeneration cycle was considered one analysis cycle. Various antibodies were measured according to this cycle. The resulting sensorgrams were analyzed for velocity using Biacore's dedicated data analysis software, Biacore T100 Evaluation Software (GE Healthcare Bioscience).

[Reference Example 4]

Method for evaluating the IL-6 receptor neutralizing activity of anti-human IL-6 receptor antibodies using BaF/6R cells

To obtain a cell line exhibiting IL-6-dependent proliferation, a BaF3 cell line expressing human gp130 and human IL-6R was established as follows. The full-length human IL-6R cDNA was amplified by PCR and cloned into pcDNA3.1(+) (Invitrogen) to construct hIL-6R/pcDNA3.1(+). The pCOS2Zeo/gp130 gene was introduced into BaF3 cells by electroporation and screened in the presence of human interleukin-6 (R&D Systems) and 100 ng/mL human interleukin-6 soluble receptor (R&D Systems) to establish a human gp130-expressing BaF3 cell line (hereinafter referred to as BaF/gp130). The full-length human IL-6R cDNA was then amplified by PCR and cloned into pcDNA3.1(+) (Invitrogen) to construct hIL-6R/pcDNA3.1(+). The pcDNA3.1(+)/hIL-6R gene was introduced into the BaF/gp130 cells prepared above by electroporation, and selection was performed in the presence of human interleukin-6 (R&D Systems) to establish the human IL-6R-expressing BaF3 cell line (hereinafter referred to as BaF/6R). This BaF/6R cell line proliferates in the presence of human interleukin-6 (R&D Systems) and can be used to evaluate the proliferation-inhibiting activity (i.e., human IL-6 receptor-neutralizing activity) of anti-human IL-6 receptor antibodies.

The human IL-6 receptor neutralizing activity of anti-human IL-6 receptor antibodies was evaluated using BaF/6R. BaF/6R was washed three times with RPMI1640 medium containing 10% FBS, then suspended in RPMI1640 medium containing 20 ng/mL human interleukin-6 (TORAY) (final concentration 10 ng/mL) and 10% FBS to a concentration of 2.5 to 5.0 × ¹⁰⁴ cells/mL. 50 μL of the suspension was then injected into each well of a 96-well plate (CORNING). Next, the anti-human IL-6 receptor antibody was diluted in RPMI1640 containing 10% FBS, and 50 μL of the suspension was mixed into each well. After culturing for 3 days at 37°C and 5% _CO₂ , 20 μL/well of WST-8 reagent (Cell Counting Kit-8, Dojindo Chemical Laboratories, Ltd.) diluted 2-fold with PBS was added. Immediately thereafter, the absorbance at 450 nm (reference wavelength: 620 nm) was measured using a SUNRISE CLASSIC (TECAN). After culturing for 2 hours, the absorbance at 450 nm (reference wavelength: 620 nm) was measured again. The change in absorbance over 2 to 4 hours was used as an indicator to evaluate the human IL-6 receptor neutralizing activity.

[Reference Example 5]

Evaluation of the binding activity of each mutant-modified anti-human GPC3 antibody by competitive ELISA

The binding activity of the prepared antibodies was evaluated by competitive ELISA. 100 μL of soluble GPC3 core polypeptide (SEQ ID NO::207) prepared at 1 μg/mL was added to each well of a 96-well plate. The plate was left to stand overnight at 4°C to immobilize the soluble GPC3 core polypeptide on the plate. The soluble GPC3 core polypeptide immobilized on the plate was washed three times with wash buffer using a Skan WASHER 400 (Molecular Devices), and 200 μL of blocking buffer was added, followed by blocking at 4°C for at least 30 minutes. Next, the plate with the immobilized and blocked soluble GPC3 core polypeptide was washed three times with wash buffer using a Skan WASHER 400. Thereafter, 200 μL of a mixture containing 100 μL of various concentrations of GPC3-H2L2 antibody or other antibody and 100 μL of biotinylated GPC3-H2L2 antibody at a final concentration of 0.3 μg/mL was added to each well of the plate. Biotinylation of the GPC3-H2L2 antibody was performed using a biotin labeling kit (Roche) according to the kit's instructions. The plate was allowed to stand at room temperature for 1 hour, then washed 5 times with wash buffer using a Skan WASHER400 (Molecular Devices). 100 μL of goat anti-streptavidin alkaline phosphatase (ZYMED) diluted to 20,000 times with matrix buffer was added to each well of the plate, the plate was allowed to stand at room temperature for 1 hour, then washed 5 times with wash buffer using a Skan WASHER400. Phosphatase substrate (Sigma) was prepared using matrix buffer to a concentration of 1 mg/mL, 100 μL was added to each well, and allowed to stand for 1 hour. Benchmark Plus (BIO-RAD) was used to measure the absorbance of the reaction solution in each well at 405 nm using a control absorbance of 655 nm.

[Reference Example 6]

Method for evaluating the affinity of anti-human IL-31 receptor antibodies to IL-31 receptor using Biacore

1. Preparation of Soluble Human IL-31 Receptor

Using the cDNA of the human IL31 receptor as a template, only the extracellular region was amplified by PCR, and a FLAG tag sequence was added to the C-terminus and integrated into an expression vector for mammalian cells. 10 μg of the linear vector was introduced into the Chinese hamster ovary cell line DG44 by electroporation (BioRad Gene PulserII, 25 μF, 1.5 kV) to obtain a highly expressing cell line. The cell line was cultured in large quantities, and soluble NR10 was purified from the culture supernatant using an anti-FLAG antibody column (manufactured by SIGMA) and gel filtration. The amino acid sequence of the soluble human IL31 receptor is shown in SEQ ID NO: 246.

2. Evaluation of affinity for soluble human IL-31 receptor using Biacore

Using Biacore T100 (GE Healthcare Bioscience), the rate of antigen-antibody reaction between anti-human IL-31 receptor antibodies and soluble human IL-31 receptor was analyzed. According to methods known to those skilled in the art, rec-Protein A (ZYMED) (hereinafter referred to as Protein A) was immobilized on a sensor chip, the antibody was captured on the immobilized Protein A, and the antigen was reacted as the analyte to measure the interaction between the antibody and the antigen. Each antibody was prepared and an appropriate amount of the antibody was bound to Protein A/G. The soluble human IL-31 receptor used as the analyte was prepared with HBS-EP+ at 0, 38.5, 77.0, and 154 nM. During the measurement, the antibody solution was first bound to Protein A/G, and the analyte solution was allowed to interact with it for 3 minutes. After that, HBS-EP+ was switched and the dissociation phase was measured for 5 minutes. After the dissociation phase measurement was completed, the sensor chip was washed with 10 μL of 10 mM glycine-HCl (pH 1.5) to regenerate the sensor chip. This binding, dissociation, and regeneration constitutes one analysis cycle. Various antibodies were measured according to this cycle. The resulting sensorgrams were analyzed kinetically using Biacore-specific data analysis software, namely Biacore T100 Evaluation Software (GE Healthcare Bioscience).

Industrial Applicability

The present invention provides methods for altering the isoelectric point of an antibody by modifying the charge of at least one amino acid residue exposed on the surface of a complementarity determining region (CDR) while maintaining antigen-binding activity, methods for purifying multispecific antibodies, methods for improving the plasma pharmacokinetics of antibodies, pharmaceutical compositions containing antibodies with altered isoelectric points as active ingredients, and methods for producing the same. By modifying the isoelectric point of an antibody, multispecific antibodies can be purified efficiently and with high purity. Furthermore, by modifying the isoelectric point of an antibody, plasma pharmacokinetics can be improved, allowing for reduced dosing frequency and sustained therapeutic effects. It should be noted that the antibodies obtained according to the methods of the present invention maintain antigen-binding activity.

<110> Chugai Pharmaceutical Co., Ltd.

<120> Method for changing the isoelectric point of an antibody by using CDR amino acid substitution

<130> C1-A0708P2

<150> JP 2007-250165

<151> 2007-09-26

<150> JP 2007-256063

<151> 2007-09-28

<160> 246

<170> PatentIn version 3.4

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<213> House Mouse

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Ser Asp His Ala Trp Ser

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Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu Lys Ser

1 5 10 15

<210> 3

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Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr

1 5 10

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Arg Ala Ser Gln Asp Ile Ser Ser Tyr Leu Asn

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<400> 5

Tyr Thr Ser Arg Leu His Ser

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Gln Gln Gly Asn Thr Leu Pro Tyr Thr

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<213> Homo sapiens

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Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln

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Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr

20 25 30

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Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp Ile Gly

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<210> 9

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<212> PRT

<213> Homo sapiens

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Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser Leu Arg

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Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg

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Trp Gly Gln Gly Ser Leu Val Thr Val Ser Ser

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Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys

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Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

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Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

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Phe Thr Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys

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Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

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<223> Artificially synthesized peptide sequence

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Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp

20 25 30

His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp

35 40 45

Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu

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Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser

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Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys

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Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gln Gly

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Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

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Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

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Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

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Asn Ser Gly Ala Leu Thr Ser Ser Gly Val His Thr Phe Pro Ala Val Leu

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Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

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Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

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Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

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Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

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Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

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Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

<210> 16

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 16

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 17

<211> 119

<212> PRT

<213> Artificial sequence

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<400> 17

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp

20 25 30

His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp

35 40 45

Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gln Gly

100 105 110

Ser Leu Val Thr Val Ser Ser

115

<210> 18

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 18

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 19

<211> 330

<212> PRT

<213> Homo sapiens

<400> 19

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

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Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

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Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

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Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

225 230 235 240

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210> 20

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<212> PRT

<213> Homo sapiens

<400> 20

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro

100 105 110

Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

115 120 125

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

130 135 140

Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly

145 150 155 160

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn

165 170 175

Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp

180 185 190

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro

195 200 205

Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu

210 215 220

Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn

225 230 235 240

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

245 250 255

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

260 265 270

Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

275 280 285

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

290 295 300

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

305 310 315 320

Ser Leu Ser Pro Gly Lys

325

<210> 21

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<213> Homo sapiens

<400> 21

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro

100 105 110

Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

115 120 125

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

130 135 140

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

145 150 155 160

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

165 170 175

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

180 185 190

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

195 200 205

Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

210 215 220

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys

225 230 235 240

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

245 250 255

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

260 265 270

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

275 280 285

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

290 295 300

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

305 310 315 320

Leu Ser Leu Ser Leu Gly Lys

325

<210> 22

<211> 119

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<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 22

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Asp Asp

20 25 30

His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp

35 40 45

Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu

50 55 60

Lys Gly Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Ala Tyr Tyr Cys

85 90 95

Ala Arg Val Leu Ala Arg Ile Thr Ala Met Asp Tyr Trp Gly Glu Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 23

<211> 107

<212> PRT

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<220>

<223> Artificially synthesized peptide sequence

<400> 23

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Ser Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Glu Leu Leu Ile

35 40 45

Tyr Tyr Gly Ser Glu Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Glu Ala

65 70 75 80

Glu Asp Ala Ala Thr Tyr Tyr Cys Gly Gln Gly Asn Arg Leu Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Glu

100 105

<210> 24

<211> 324

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<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 24

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Thr Val Glu Arg Lys Ser Cys Val Glu Cys Pro Pro Cys Pro Ala Pro

100 105 110

Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

115 120 125

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

130 135 140

Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly

145 150 155 160

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn

165 170 175

Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp

180 185 190

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro

195 200 205

Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu

210 215 220

Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn

225 230 235 240

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

245 250 255

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

260 265 270

Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

275 280 285

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

290 295 300

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

305 310 315 320

Ser Leu Ser Pro

<210> 25

<211> 324

<212> PRT

<213> Artificial sequence

<220>

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<400> 25

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Thr Val Glu Arg Lys Ser Cys Val Glu Cys Pro Pro Cys Pro Ala Pro

100 105 110

Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

115 120 125

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

130 135 140

Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly

145 150 155 160

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn

165 170 175

Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp

180 185 190

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro

195 200 205

Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu

210 215 220

Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn

225 230 235 240

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

245 250 255

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

260 265 270

Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

275 280 285

Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys

290 295 300

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

305 310 315 320

Ser Leu Ser Leu

<210> 26

<211> 6

<212> PRT

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<400> 26

Trp Asp His Ala Trp Ser

1 5

<210> 27

<211> 6

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<220>

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<400> 27

Thr Asp His Ala Trp Ser

1 5

<210> 28

<211> 6

<212> PRT

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<220>

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<400> 28

Asp Asp His Ala Trp Ser

1 5

<210> 29

<211> 6

<212> PRT

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<220>

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<400> 29

Asn Asp His Ala Trp Ser

1 5

<210> 30

<211> 6

<212> PRT

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<220>

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<400> 30

Arg Asp His Ala Trp Ser

1 5

<210> 31

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 31

Val Asp His Ala Trp Ser

1 5

<210> 32

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 32

Phe Asp His Ala Trp Ser

1 5

<210> 33

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 33

Ala Asp His Ala Trp Ser

1 5

<210> 34

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 34

Gln Asp His Ala Trp Ser

1 5

<210> 35

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 35

Tyr Asp His Ala Trp Ser

1 5

<210> 36

<211> 6

<212> PRT

<213> Artificial sequence

<220>

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<400> 36

Leu Asp His Ala Trp Ser

1 5

<210> 37

<211> 6

<212> PRT

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<220>

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<400> 37

His Asp His Ala Trp Ser

1 5

<210> 38

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 38

Glu Asp His Ala Trp Ser

1 5

<210> 39

<211> 6

<212> PRT

<213> Artificial sequence

<220>

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<400> 39

Cys Asp His Ala Trp Ser

1 5

<210> 40

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 40

Ser Asp His Ala Ile Ser

1 5

<210> 41

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 41

Ser Asp His Ala Val Ser

1 5

<210> 42

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 42

Phe Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu Lys Ser

1 5 10 15

<210> 43

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 43

Tyr Ile Ser Tyr Ser Gly Ile Arg Thr Tyr Asn Pro Ser Leu Lys Ser

1 5 10 15

<210> 44

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 44

Tyr Ile Ser Tyr Ser Gly Ile Thr Ser Tyr Asn Pro Ser Leu Lys Ser

1 5 10 15

<210> 45

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 45

Tyr Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu Lys Ser

1 5 10 15

<210> 46

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 46

Ile Leu Ala Arg Thr Thr Ala Met Asp Tyr

1 5 10

<210> 47

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 47

Val Leu Ala Arg Thr Thr Ala Met Asp Tyr

1 5 10

<210> 48

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 48

Thr Leu Ala Arg Thr Thr Ala Met Asp Tyr

1 5 10

<210> 49

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 49

Leu Leu Ala Arg Thr Thr Ala Met Asp Tyr

1 5 10

<210> 50

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 50

Ser Thr Ala Arg Thr Thr Ala Met Asp Tyr

1 5 10

<210> 51

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 51

Ser Leu Ala Arg Ala Thr Ala Met Asp Tyr

1 5 10

<210> 52

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 52

Ser Leu Ala Arg Ile Thr Ala Met Asp Tyr

1 5 10

<210> 53

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 53

Ser Leu Ala Arg Ser Thr Ala Met Asp Tyr

1 5 10

<210> 54

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 54

Ser Leu Ala Arg Thr Thr Ser Met Asp Tyr

1 5 10

<210> 55

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 55

Ser Leu Ala Arg Thr Thr Val Met Asp Tyr

1 5 10

<210> 56

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 56

Ser Leu Ala Arg Thr Thr Ala Leu Asp Tyr

1 5 10

<210> 57

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 57

Leu Leu Ala Arg Ala Thr Ala Met Asp Tyr

1 5 10

<210> 58

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 58

Val Leu Ala Arg Ala Thr Ala Met Asp Tyr

1 5 10

<210> 59

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 59

Ile Leu Ala Arg Ala Thr Ala Met Asp Tyr

1 5 10

<210> 60

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 60

Thr Leu Ala Arg Ala Thr Ala Met Asp Tyr

1 5 10

<210> 61

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 61

Val Leu Ala Arg Ile Thr Ala Met Asp Tyr

1 5 10

<210> 62

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 62

Ile Leu Ala Arg Ile Thr Ala Met Asp Tyr

1 5 10

<210> 63

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 63

Thr Leu Ala Arg Ile Thr Ala Met Asp Tyr

1 5 10

<210> 64

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 64

Leu Leu Ala Arg Ile Thr Ala Met Asp Tyr

1 5 10

<210> 65

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 65

Ser Thr Ala Arg Thr Thr Val Leu Asp Tyr

1 5 10

<210> 66

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 66

Phe Ala Ser Gln Asp Ile Ser Ser Tyr Leu Asn

1 5 10

<210> 67

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 67

Arg Ala Ser Arg Asp Ile Ser Ser Tyr Leu Asn

1 5 10

<210> 68

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 68

Arg Ala Ser Thr Asp Ile Ser Ser Tyr Leu Asn

1 5 10

<210> 69

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 69

Arg Ala Ser Gln Asp Ile Ser Ser Phe Leu Asn

1 5 10

<210> 70

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 70

Arg Ala Ser Gln Asp Ile Ser Ser Tyr Leu Ser

1 5 10

<210> 71

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 71

Tyr Gly Ser Arg Leu His Ser

1 5

<210> 72

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 72

Gly Gln Gly Asn Thr Leu Pro Tyr Thr

1 5

<210> 73

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 73

Asn Gln Gly Asn Thr Leu Pro Tyr Thr

1 5

<210> 74

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 74

Ser Gln Gly Asn Thr Leu Pro Tyr Thr

1 5

<210> 75

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 75

Gln Gln Ser Asn Thr Leu Pro Tyr Thr

1 5

<210> 76

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 76

Gln Gln Gly Asn Arg Leu Pro Tyr Thr

1 5

<210> 77

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 77

Gln Gln Gly Asn Ser Leu Pro Tyr Thr

1 5

<210> 78

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 78

Gly Gln Gly Asn Ser Leu Pro Tyr Thr

1 5

<210> 79

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 79

Gly Gln Gly Asn Arg Leu Pro Tyr Thr

1 5

<210> 80

<211> 30

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 80

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr

20 25 30

<210> 81

<211> 30

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 81

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr

20 25 30

<210> 82

<211> 30

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 82

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Thr

20 25 30

<210> 83

<211> 30

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 83

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Ser

20 25 30

<210> 84

<211> 30

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 84

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser

20 25 30

<210> 85

<211> 14

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 85

Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp Ile Gly

1 5 10

<210> 86

<211> 32

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 86

Arg Val Thr Ile Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser Leu Arg

1 5 10 15

Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg

20 25 30

<210> 87

<211> 32

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 87

Arg Val Thr Met Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser Leu Arg

1 5 10 15

Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg

20 25 30

<210> 88

<211> 32

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 88

Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser Leu Lys

1 5 10 15

Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg

20 25 30

<210> 89

<211> 32

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 89

Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser Leu Arg

1 5 10 15

Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Ala Tyr Tyr Cys Ala Arg

20 25 30

<210> 90

<211> 32

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 90

Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser Leu Lys

1 5 10 15

Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Ala Tyr Tyr Cys Ala Arg

20 25 30

<210> 91

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 91

Trp Gly Glu Gly Ser Leu Val Thr Val Ser Ser

1 5 10

<210> 92

<211> 23

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 92

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Ser Val Thr Ile Thr Cys

20

<210> 93

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 93

Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Glu Leu Leu Ile Tyr

1 5 10 15

<210> 94

<211> 32

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 94

Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

1 5 10 15

Phe Thr Ile Ser Ser Leu Glu Pro Glu Asp Ile Ala Thr Tyr Tyr Cys

20 25 30

<210> 95

<211> 32

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 95

Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

1 5 10 15

Phe Thr Ile Ser Ser Leu Gln Ala Glu Asp Ile Ala Thr Tyr Tyr Cys

20 25 30

<210> 96

<211> 32

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 96

Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

1 5 10 15

Phe Thr Ile Ser Ser Leu Gln Pro Glu Asp Ala Ala Thr Tyr Tyr Cys

20 25 30

<210> 97

<211> 32

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 97

Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

1 5 10 15

Phe Thr Ile Ser Ser Leu Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys

20 25 30

<210> 98

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 98

Phe Gly Gln Gly Thr Lys Val Glu Ile Glu

1 5 10

<210> 99

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 99

Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu Lys Gly

1 5 10 15

<210> 100

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 100

Tyr Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu Lys Gly

1 5 10 15

<210> 101

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 101

Gln Ala Ser Gln Asp Ile Ser Ser Tyr Leu Asn

1 5 10

<210> 102

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 102

Tyr Thr Ser Glu Leu His Ser

1 5

<210> 103

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 103

Tyr Gly Ser Glu Leu His Ser

1 5

<210> 104

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 104

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Asp Asp

20 25 30

His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp

35 40 45

Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu

50 55 60

Lys Gly Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Ala Tyr Tyr Cys

85 90 95

Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 105

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 105

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Ser Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Glu Leu Leu Ile

35 40 45

Tyr Tyr Gly Ser Glu Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Glu Ala

65 70 75 80

Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Ser Leu Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Glu

100 105

<210> 106

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 106

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 107

<211> 414

<212> DNA

<213> Artificial sequence

<220>

<223> Artificially synthesized nucleotide sequences

<400> 107

atgggatgga gctgtatcat cctcttcttg gtagcaacag ctacaggtgt ccactcccag 60

gtccaactgc aggagagcgg tccaggtctt gtgagaccta gccagaccct gagcctgacc 120

tgcaccgtgt ctggctactc aattaccagc gatcatgcct ggagctgggt tcgccagcca 180

cctggacgag gtcttgagtg gattggatac attagttata gtggaatcac aacctataat 240

ccatctctca aatccagagt gacaatgctg agagacacca gcaagaacca gttcagcctg 300

agactcagca gcgtgacagc cgccgacacc gcggtttat attgtgcaag atccctagct 360

cggactacgg ctatggacta ctggggtcaa ggcagcctcg tcacagtctc ctca 414

<210> 108

<211> 378

<212> DNA

<213> Artificial sequence

<220>

<223> Artificially synthesized nucleotide sequences

<400> 108

atgggatgga gctgtatcat cctcttcttg gtagcaacag ctacaggtgt ccactccgac 60

atccagatga cccagagccc aagcagcctg agcgccagcg tgggtgacag agtgaccatc 120

acctgtagag ccagccagga catcagcagt tacctgaatt ggtaccagca gaagccagga 180

aaggctccaa agctgctgat ctactacacc tccagactgc actctggtgt gccaagcaga 240

ttcagcggta gcggtagcgg taccgacttc accttcacca tcagcagcct ccagccagag 300

gacatcgcta cctactactg ccaacagggt aacacgcttc catacacgtt cggccaaggg 360

accaaggtgg aaatcaaa 378

<210> 109

<211> 445

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 109

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp

20 25 30

His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp

35 40 45

Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gln Gly

100 105 110

Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys Cys Val Glu

210 215 220

Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu

225 230 235 240

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

245 250 255

Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln

260 265 270

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

275 280 285

Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu

290 295 300

Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

305 310 315 320

Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys

325 330 335

Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

340 345 350

Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

355 360 365

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

370 375 380

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly

385 390 395 400

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

405 410 415

Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

420 425 430

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 110

<211> 446

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 110

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp

20 25 30

His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp

35 40 45

Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gln Gly

100 105 110

Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro

210 215 220

Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe

225 230 235 240

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

245 250 255

Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val

260 265 270

Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

275 280 285

Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val

290 295 300

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

305 310 315 320

Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser

325 330 335

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

340 345 350

Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

355 360 365

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

370 375 380

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

385 390 395 400

Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp

405 410 415

Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

420 425 430

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440 445

<210> 111

<211> 445

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 111

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp

20 25 30

His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp

35 40 45

Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gln Gly

100 105 110

Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys Cys Val Glu

210 215 220

Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu

225 230 235 240

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

245 250 255

Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln

260 265 270

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

275 280 285

Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu

290 295 300

Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

305 310 315 320

Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys

325 330 335

Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

340 345 350

Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

355 360 365

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

370 375 380

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

385 390 395 400

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

405 410 415

Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

420 425 430

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 112

<211> 446

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 112

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp

20 25 30

His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp

35 40 45

Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gln Gly

100 105 110

Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro

210 215 220

Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe

225 230 235 240

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

245 250 255

Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val

260 265 270

Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

275 280 285

Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val

290 295 300

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

305 310 315 320

Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser

325 330 335

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

340 345 350

Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

355 360 365

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

370 375 380

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

385 390 395 400

Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

405 410 415

Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

420 425 430

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440 445

<210> 113

<211> 443

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 113

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp

20 25 30

His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp

35 40 45

Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gln Gly

100 105 110

Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Ser Cys Val Glu

210 215 220

Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu

225 230 235 240

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

245 250 255

Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln

260 265 270

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

275 280 285

Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu

290 295 300

Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

305 310 315 320

Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys

325 330 335

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

340 345 350

Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

355 360 365

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

370 375 380

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

385 390 395 400

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

405 410 415

Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

420 425 430

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440

<210> 114

<211> 443

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 114

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp

20 25 30

His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp

35 40 45

Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gln Gly

100 105 110

Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Ser Cys Val Glu

210 215 220

Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu

225 230 235 240

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

245 250 255

Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln

260 265 270

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

275 280 285

Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu

290 295 300

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

305 310 315 320

Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys

325 330 335

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

340 345 350

Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

355 360 365

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

370 375 380

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

385 390 395 400

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

405 410 415

Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

420 425 430

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu

435 440

<210> 115

<211> 446

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 115

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp

20 25 30

His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp

35 40 45

Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gln Gly

100 105 110

Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

405 410 415

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

<210> 116

<211> 327

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 116

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

115 120 125

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

130 135 140

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

145 150 155 160

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

165 170 175

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

180 185 190

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

195 200 205

Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

210 215 220

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu

225 230 235 240

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

245 250 255

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

260 265 270

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

275 280 285

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val

290 295 300

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

305 310 315 320

Lys Ser Leu Ser Leu Ser Pro

325

<210> 117

<211> 443

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 117

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Asp Asp

20 25 30

His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp

35 40 45

Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu

50 55 60

Lys Gly Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Ala Tyr Tyr Cys

85 90 95

Ala Arg Val Leu Ala Arg Ile Thr Ala Met Asp Tyr Trp Gly Glu Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Ser Cys Val Glu

210 215 220

Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu

225 230 235 240

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

245 250 255

Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln

260 265 270

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

275 280 285

Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu

290 295 300

Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

305 310 315 320

Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys

325 330 335

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

340 345 350

Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

355 360 365

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

370 375 380

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

385 390 395 400

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

405 410 415

Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

420 425 430

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440

<210> 118

<211> 324

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 118

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Thr Val Glu Arg Lys Ser Cys Val Glu Cys Pro Pro Cys Pro Ala Pro

100 105 110

Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

115 120 125

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

130 135 140

Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly

145 150 155 160

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn

165 170 175

Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp

180 185 190

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro

195 200 205

Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu

210 215 220

Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn

225 230 235 240

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

245 250 255

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

260 265 270

Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

275 280 285

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

290 295 300

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

305 310 315 320

Ser Leu Ser Pro

<210> 119

<211> 443

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 119

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp

20 25 30

His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp

35 40 45

Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gln Gly

100 105 110

Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Ser Cys Val Glu

210 215 220

Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu

225 230 235 240

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

245 250 255

Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln

260 265 270

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

275 280 285

Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu

290 295 300

Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

305 310 315 320

Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys

325 330 335

Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

340 345 350

Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

355 360 365

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

370 375 380

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

385 390 395 400

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

405 410 415

Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

420 425 430

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440

<210> 120

<211> 445

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 120

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp

20 25 30

His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp

35 40 45

Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gln Gly

100 105 110

Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Glu Ser Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Ser Cys Val Glu

210 215 220

Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu

225 230 235 240

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

245 250 255

Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln

260 265 270

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

275 280 285

Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu

290 295 300

Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

305 310 315 320

Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys

325 330 335

Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

340 345 350

Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

355 360 365

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

370 375 380

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly

385 390 395 400

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

405 410 415

Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

420 425 430

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 121

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 121

Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu Gln Asp

1 5 10 15

<210> 122

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 122

Tyr Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu Gln Asp

1 5 10 15

<210> 123

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 123

Arg Ala Ser Glu Asp Ile Ser Ser Tyr Leu Asn

1 5 10

<210> 124

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 124

Gln Ala Ser Glu Asp Ile Ser Ser Tyr Leu Asn

1 5 10

<210> 125

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 125

Tyr Thr Ser Arg Leu Glu Ser

1 5

<210> 126

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 126

Tyr Gly Ser Glu Leu Glu Ser

1 5

<210> 127

<211> 32

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 127

Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser Leu Lys

1 5 10 15

Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg

20 25 30

<210> 128

<211> 32

<212> PRT

<213> Homo sapiens

<400> 128

Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln

1 5 10 15

Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg

20 25 30

<210> 129

<211> 32

<212> PRT

<213> Homo sapiens

<400> 129

Arg Val Thr Ile Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr Met Glu

1 5 10 15

Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg

20 25 30

<210> 130

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 130

Trp Gly Gln Gly Ile Leu Val Thr Val Ser Ser

1 5 10

<210> 131

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 131

Trp Gly Glu Gly Ile Leu Val Thr Val Ser Ser

1 5 10

<210> 132

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 132

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

1 5 10

<210> 133

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 133

Trp Gly Glu Gly Thr Leu Val Thr Val Ser Ser

1 5 10

<210> 134

<211> 449

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 134

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Asp Asp

20 25 30

His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp

35 40 45

Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu

50 55 60

Lys Gly Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Val Leu Ala Arg Ile Thr Ala Met Asp Tyr Trp Gly Glu Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

Lys

<210> 135

<211> 449

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 135

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Asp Asp

20 25 30

His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp

35 40 45

Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu

50 55 60

Gln Asp Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Leu Leu Ala Arg Ala Thr Ala Met Asp Tyr Trp Gly Gln Gly

100 105 110

Ile Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

Lys

<210> 136

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 136

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Ser Val Thr Ile Thr Cys Gln Ala Ser Glu Asp Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Glu Leu Leu Ile

35 40 45

Tyr Tyr Gly Ser Glu Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Glu Ala

65 70 75 80

Glu Asp Ala Ala Thr Tyr Tyr Cys Gly Gln Gly Asn Arg Leu Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Glu Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 137

<211> 449

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 137

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Asp Asp

20 25 30

His Ala Val Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp

35 40 45

Ile Gly Phe Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Thr Leu

50 55 60

Gln Asp Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Leu Leu Ala Arg Ala Thr Ala Met Asp Val Trp Gly Glu Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

Lys

<210> 138

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 138

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Ser Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Glu Leu Leu Ile

35 40 45

Tyr Tyr Gly Ser Glu Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Glu Ala

65 70 75 80

Glu Asp Ala Ala Thr Tyr Tyr Cys Gly Gln Gly Asn Arg Leu Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Glu Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 139

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 139

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Asp Asp

20 25 30

His Ala Val Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp

35 40 45

Ile Gly Phe Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Thr Leu

50 55 60

Gln Asp Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Leu Leu Ala Arg Ala Thr Ala Met Asp Val Trp Gly Glu Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Ala His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

<210> 140

<211> 267

<212> PRT

<213> Homo sapiens

<400> 140

Ala Glu Ser His Leu Ser Leu Leu Tyr His Leu Thr Ala Val Ser Ser

1 5 10 15

Pro Ala Pro Gly Thr Pro Ala Phe Trp Val Ser Gly Trp Leu Gly Pro

20 25 30

Gln Gln Tyr Leu Ser Tyr Asn Ser Leu Arg Gly Glu Ala Glu Pro Cys

35 40 45

Gly Ala Trp Val Trp Glu Asn Gln Val Ser Trp Tyr Trp Glu Lys Glu

50 55 60

Thr Thr Asp Leu Arg Ile Lys Glu Lys Leu Phe Leu Glu Ala Phe Lys

65 70 75 80

Ala Leu Gly Gly Lys Gly Pro Tyr Thr Leu Gln Gly Leu Leu Gly Cys

85 90 95

Glu Leu Gly Pro Asp Asn Thr Ser Val Pro Thr Ala Lys Phe Ala Leu

100 105 110

Asn Gly Glu Glu Phe Met Asn Phe Asp Leu Lys Gln Gly Thr Trp Gly

115 120 125

Gly Asp Trp Pro Glu Ala Leu Ala Ile Ser Gln Arg Trp Gln Gln Gln

130 135 140

Asp Lys Ala Ala Asn Lys Glu Leu Thr Phe Leu Leu Phe Ser Cys Pro

145 150 155 160

His Arg Leu Arg Glu His Leu Glu Arg Gly Arg Gly Asn Leu Glu Trp

165 170 175

Lys Glu Pro Pro Ser Met Arg Leu Lys Ala Arg Pro Ser Ser Pro Gly

180 185 190

Phe Ser Val Leu Thr Cys Ser Ala Phe Ser Phe Tyr Pro Pro Glu Leu

195 200 205

Gln Leu Arg Phe Leu Arg Asn Gly Leu Ala Ala Gly Thr Gly Gln Gly

210 215 220

Asp Phe Gly Pro Asn Ser Asp Gly Ser Phe His Ala Ser Ser Ser Leu

225 230 235 240

Thr Val Lys Ser Gly Asp Glu His His Tyr Cys Cys Ile Val Gln His

245 250 255

Ala Gly Leu Ala Gln Pro Leu Arg Val Glu Leu

260 265

<210> 141

<211> 99

<212> PRT

<213> Homo sapiens

<400> 141

Ile Gln Arg Thr Pro Lys Ile Gln Val Tyr Ser Arg His Pro Ala Glu

1 5 10 15

Asn Gly Lys Ser Asn Phe Leu Asn Cys Tyr Val Ser Gly Phe His Pro

20 25 30

Ser Asp Ile Glu Val Asp Leu Leu Lys Asn Gly Glu Arg Ile Glu Lys

35 40 45

Val Glu His Ser Asp Leu Ser Phe Ser Lys Asp Trp Ser Phe Tyr Leu

50 55 60

Leu Tyr Tyr Thr Glu Phe Thr Pro Thr Glu Lys Asp Glu Tyr Ala Cys

65 70 75 80

Arg Val Asn His Val Thr Leu Ser Gln Pro Lys Ile Val Lys Trp Asp

85 90 95

Arg Asp Met

<210> 142

<211> 443

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 142

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp

20 25 30

His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp

35 40 45

Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gln Gly

100 105 110

Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Ser Cys Val Glu

210 215 220

Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu

225 230 235 240

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

245 250 255

Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln

260 265 270

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

275 280 285

Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu

290 295 300

Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

305 310 315 320

Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys

325 330 335

Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

340 345 350

Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

355 360 365

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

370 375 380

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly

385 390 395 400

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

405 410 415

Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

420 425 430

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440

<210> 143

<211> 449

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 143

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp

20 25 30

His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp

35 40 45

Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gln Gly

100 105 110

Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Ala His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

Lys

<210> 144

<211> 443

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 144

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp

20 25 30

His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp

35 40 45

Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gln Gly

100 105 110

Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Ser Cys Val Glu

210 215 220

Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu

225 230 235 240

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

245 250 255

Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln

260 265 270

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

275 280 285

Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu

290 295 300

Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

305 310 315 320

Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys

325 330 335

Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

340 345 350

Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

355 360 365

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

370 375 380

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly

385 390 395 400

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

405 410 415

Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Ala

420 425 430

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440

<210> 145

<211> 119

<212> PRT

<213> House Mouse

<400> 145

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Asp Asp

20 25 30

His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp

35 40 45

Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu

50 55 60

Gln Asp Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Leu Leu Ala Arg Ala Thr Ala Met Asp Tyr Trp Gly Gln Gly

100 105 110

Ile Leu Val Thr Val Ser Ser

115

<210> 146

<211> 107

<212> PRT

<213> House Mouse

<400> 146

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Ser Val Thr Ile Thr Cys Gln Ala Ser Glu Asp Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Glu Leu Leu Ile

35 40 45

Tyr Tyr Gly Ser Glu Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Glu Ala

65 70 75 80

Glu Asp Ala Ala Thr Tyr Tyr Cys Gly Gln Gly Asn Arg Leu Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Glu

100 105

<210> 147

<211> 120

<212> PRT

<213> House Mouse

<400> 147

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr

20 25 30

Ile Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Leu Ile Asn Pro Tyr Asn Gly Gly Thr Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Gly Tyr Asp Asp Gly Pro Tyr Thr Met Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser

115 120

<210> 148

<211> 107

<212> PRT

<213> House Mouse

<400> 148

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Thr Ser Glu Asn Ile Tyr Ser Phe

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Asn Ala Lys Thr Leu Ala Lys Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His His Tyr Glu Ser Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 149

<211> 122

<212> PRT

<213> House Mouse

<400> 149

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Gly Ile Thr Gly Ser Gly Gly Ser Thr Tyr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Asp Pro Gly Thr Thr Val Ile Met Ser Trp Phe Asp Pro Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 150

<211> 108

<212> PRT

<213> House Mouse

<400> 150

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Arg Gly Arg

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Phe Tyr Cys Gln Gln Tyr Gly Ser Ser Pro

85 90 95

Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 151

<211> 324

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 151

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Thr Val Glu Arg Lys Ser Cys Val Glu Cys Pro Pro Cys Pro Ala Pro

100 105 110

Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

115 120 125

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

130 135 140

Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly

145 150 155 160

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn

165 170 175

Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp

180 185 190

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro

195 200 205

Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu

210 215 220

Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn

225 230 235 240

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

245 250 255

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

260 265 270

Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

275 280 285

Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys

290 295 300

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

305 310 315 320

Ser Leu Ser Pro

<210> 152

<211> 330

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 152

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

225 230 235 240

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Ala His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210> 153

<211> 324

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 153

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Thr Val Glu Arg Lys Ser Cys Val Glu Cys Pro Pro Cys Pro Ala Pro

100 105 110

Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

115 120 125

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

130 135 140

Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly

145 150 155 160

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn

165 170 175

Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp

180 185 190

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro

195 200 205

Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu

210 215 220

Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn

225 230 235 240

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

245 250 255

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

260 265 270

Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

275 280 285

Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys

290 295 300

Ser Val Met His Glu Ala Leu His Ala His Tyr Thr Gln Lys Ser Leu

305 310 315 320

Ser Leu Ser Pro

<210> 154

<211> 326

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 154

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Thr Val Glu Arg Lys Ser Cys Val Glu Cys Pro Pro Cys Pro Ala Pro

100 105 110

Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

115 120 125

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

130 135 140

Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly

145 150 155 160

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn

165 170 175

Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp

180 185 190

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro

195 200 205

Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu

210 215 220

Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn

225 230 235 240

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

245 250 255

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

260 265 270

Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

275 280 285

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

290 295 300

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

305 310 315 320

Ser Leu Ser Pro Gly Lys

325

<210> 155

<211> 326

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 155

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Thr Val Glu Arg Lys Ser Cys Val Glu Cys Pro Pro Cys Pro Ala Pro

100 105 110

Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

115 120 125

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

130 135 140

Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly

145 150 155 160

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn

165 170 175

Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp

180 185 190

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro

195 200 205

Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu

210 215 220

Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn

225 230 235 240

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

245 250 255

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

260 265 270

Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

275 280 285

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

290 295 300

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

305 310 315 320

Ser Leu Ser Pro Gly Lys

325

<210> 156

<211> 326

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 156

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Thr Val Glu Arg Lys Cys Ser Val Glu Cys Pro Pro Cys Pro Ala Pro

100 105 110

Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

115 120 125

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

130 135 140

Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly

145 150 155 160

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn

165 170 175

Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp

180 185 190

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro

195 200 205

Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu

210 215 220

Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn

225 230 235 240

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

245 250 255

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

260 265 270

Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

275 280 285

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

290 295 300

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

305 310 315 320

Ser Leu Ser Pro Gly Lys

325

<210> 157

<211> 445

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 157

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp

20 25 30

His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp

35 40 45

Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gln Gly

100 105 110

Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Ser Cys Val Glu

210 215 220

Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu

225 230 235 240

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

245 250 255

Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln

260 265 270

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

275 280 285

Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu

290 295 300

Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

305 310 315 320

Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys

325 330 335

Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

340 345 350

Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

355 360 365

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

370 375 380

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly

385 390 395 400

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

405 410 415

Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

420 425 430

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 158

<211> 445

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 158

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp

20 25 30

His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp

35 40 45

Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gln Gly

100 105 110

Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys Ser Val Glu

210 215 220

Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu

225 230 235 240

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

245 250 255

Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln

260 265 270

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

275 280 285

Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu

290 295 300

Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

305 310 315 320

Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys

325 330 335

Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

340 345 350

Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

355 360 365

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

370 375 380

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly

385 390 395 400

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

405 410 415

Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

420 425 430

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 159

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 159

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Asp Asp

20 25 30

His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp

35 40 45

Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu

50 55 60

Lys Gly Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Val Leu Ala Arg Ile Thr Ala Met Asp Tyr Trp Gly Glu Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 160

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 160

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Asp Asp

20 25 30

His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp

35 40 45

Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu

50 55 60

Gln Asp Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Leu Leu Ala Arg Ala Thr Ala Met Asp Tyr Trp Gly Gln Gly

100 105 110

Ile Leu Val Thr Val Ser Ser

115

<210> 161

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 161

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Asp Asp

20 25 30

His Ala Val Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp

35 40 45

Ile Gly Phe Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Thr Leu

50 55 60

Gln Asp Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Leu Leu Ala Arg Ala Thr Ala Met Asp Val Trp Gly Glu Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 162

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 162

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Ser Val Thr Ile Thr Cys Gln Ala Ser Glu Asp Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Glu Leu Leu Ile

35 40 45

Tyr Tyr Gly Ser Glu Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Glu Ala

65 70 75 80

Glu Asp Ala Ala Thr Tyr Tyr Cys Gly Gln Gly Asn Arg Leu Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Glu

100 105

<210> 163

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 163

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Ser Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Glu Leu Leu Ile

35 40 45

Tyr Tyr Gly Ser Glu Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Glu Ala

65 70 75 80

Glu Asp Ala Ala Thr Tyr Tyr Cys Gly Gln Gly Asn Arg Leu Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Glu

100 105

<210> 164

<211> 328

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 164

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

225 230 235 240

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Ala His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro

325

<210> 165

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 165

Asp Asp His Ala Val Ser

1 5

<210> 166

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 166

Phe Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Thr Leu Gln Asp

1 5 10 15

<210> 167

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 167

Leu Leu Ala Arg Ala Thr Ala Met Asp Val

1 5 10

<210> 168

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 168

Tyr Gly Ser Glu Leu Glu Ser

1 5

<210> 169

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 169

His Asp His Ala Trp Ser

1 5

<210> 170

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 170

Phe Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu Gln Gly

1 5 10 15

<210> 171

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 171

Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr

1 5 10

<210> 172

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 172

Gln Ala Ser Thr Asp Ile Ser Ser His Leu Asn

1 5 10

<210> 173

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 173

Tyr Gly Ser His Leu Leu Ser

1 5

<210> 174

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 174

Phe Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Thr Leu Gln Gly

1 5 10 15

<210> 175

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 175

Gln Ala Ser Arg Asp Ile Ser Ser His Leu Asn

1 5 10

<210> 176

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 176

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Ser Gly His Ser Ile Ser His Asp

20 25 30

His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp

35 40 45

Ile Gly Phe Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu

50 55 60

Gln Gly Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 177

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 177

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Ser Val Thr Ile Thr Cys Gln Ala Ser Thr Asp Ile Ser Ser His

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Glu Leu Leu Ile

35 40 45

Tyr Tyr Gly Ser His Leu Leu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Glu Ala

65 70 75 80

Glu Asp Ala Ala Thr Tyr Tyr Cys Gly Gln Gly Asn Arg Leu Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Glu

100 105

<210> 178

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 178

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Ser Gly His Ser Ile Ser His Asp

20 25 30

His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp

35 40 45

Ile Gly Phe Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Thr Leu

50 55 60

Gln Gly Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 179

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 179

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Ser Val Thr Ile Thr Cys Gln Ala Ser Arg Asp Ile Ser Ser His

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Glu Leu Leu Ile

35 40 45

Tyr Tyr Gly Ser His Leu Leu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Glu Ala

65 70 75 80

Glu Asp Ala Ala Thr Tyr Tyr Cys Gly Gln Gly Asn Arg Leu Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Glu

100 105

<210> 180

<211> 443

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 180

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Ser Gly His Ser Ile Ser His Asp

20 25 30

His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp

35 40 45

Ile Gly Phe Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu

50 55 60

Gln Gly Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Ser Cys Val Glu

210 215 220

Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu

225 230 235 240

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

245 250 255

Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln

260 265 270

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

275 280 285

Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu

290 295 300

Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

305 310 315 320

Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys

325 330 335

Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

340 345 350

Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

355 360 365

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

370 375 380

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly

385 390 395 400

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

405 410 415

Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Ala

420 425 430

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440

<210> 181

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 181

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Ser Val Thr Ile Thr Cys Gln Ala Ser Thr Asp Ile Ser Ser His

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Glu Leu Leu Ile

35 40 45

Tyr Tyr Gly Ser His Leu Leu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Glu Ala

65 70 75 80

Glu Asp Ala Ala Thr Tyr Tyr Cys Gly Gln Gly Asn Arg Leu Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Glu Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 182

<211> 443

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 182

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Ser Gly His Ser Ile Ser His Asp

20 25 30

His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp

35 40 45

Ile Gly Phe Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Thr Leu

50 55 60

Gln Gly Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Ser Cys Val Glu

210 215 220

Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu

225 230 235 240

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

245 250 255

Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln

260 265 270

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

275 280 285

Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu

290 295 300

Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

305 310 315 320

Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys

325 330 335

Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

340 345 350

Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

355 360 365

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

370 375 380

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly

385 390 395 400

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

405 410 415

Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Ala

420 425 430

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440

<210> 183

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 183

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Ser Val Thr Ile Thr Cys Gln Ala Ser Arg Asp Ile Ser Ser His

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Glu Leu Leu Ile

35 40 45

Tyr Tyr Gly Ser His Leu Leu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Glu Ala

65 70 75 80

Glu Asp Ala Ala Thr Tyr Tyr Cys Gly Gln Gly Asn Arg Leu Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Glu Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 184

<211> 32

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 184

Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln

1 5 10 15

Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg

20 25 30

<210> 185

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 185

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp

20 25 30

His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp

35 40 45

Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gln Gly

100 105 110

Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Ala His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

<210> 186

<211> 30

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 186

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Ser Gly His Ser Ile Ser

20 25 30

<210> 187

<211> 445

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 187

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp

20 25 30

His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp

35 40 45

Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gln Gly

100 105 110

Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Ser Cys Val Glu

210 215 220

Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu

225 230 235 240

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

245 250 255

Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln

260 265 270

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

275 280 285

Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu

290 295 300

Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

305 310 315 320

Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys

325 330 335

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

340 345 350

Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

355 360 365

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

370 375 380

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly

385 390 395 400

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

405 410 415

Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

420 425 430

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 188

<211> 443

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 188

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp

20 25 30

His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp

35 40 45

Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gln Gly

100 105 110

Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Ser Cys Val Glu

210 215 220

Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu

225 230 235 240

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

245 250 255

Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln

260 265 270

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

275 280 285

Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu

290 295 300

Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

305 310 315 320

Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys

325 330 335

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

340 345 350

Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

355 360 365

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

370 375 380

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly

385 390 395 400

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

405 410 415

Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

420 425 430

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440

<210> 189

<211> 445

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 189

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp

20 25 30

His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp

35 40 45

Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gln Gly

100 105 110

Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Ser Cys Val Glu

210 215 220

Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu

225 230 235 240

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

245 250 255

Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln

260 265 270

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

275 280 285

Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu

290 295 300

Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

305 310 315 320

Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys

325 330 335

Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

340 345 350

Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

355 360 365

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

370 375 380

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly

385 390 395 400

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

405 410 415

Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

420 425 430

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 190

<211> 443

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 190

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp

20 25 30

His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp

35 40 45

Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gln Gly

100 105 110

Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Ser Cys Val Glu

210 215 220

Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu

225 230 235 240

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

245 250 255

Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln

260 265 270

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

275 280 285

Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu

290 295 300

Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

305 310 315 320

Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys

325 330 335

Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

340 345 350

Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

355 360 365

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

370 375 380

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly

385 390 395 400

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

405 410 415

Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

420 425 430

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440

<210> 191

<211> 326

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 191

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Thr Val Glu Arg Lys Ser Cys Val Glu Cys Pro Pro Cys Pro Ala Pro

100 105 110

Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

115 120 125

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

130 135 140

Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly

145 150 155 160

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn

165 170 175

Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp

180 185 190

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro

195 200 205

Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu

210 215 220

Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn

225 230 235 240

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

245 250 255

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

260 265 270

Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

275 280 285

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

290 295 300

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

305 310 315 320

Ser Leu Ser Pro Gly Lys

325

<210> 192

<211> 324

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 192

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Thr Val Glu Arg Lys Ser Cys Val Glu Cys Pro Pro Cys Pro Ala Pro

100 105 110

Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

115 120 125

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

130 135 140

Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly

145 150 155 160

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn

165 170 175

Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp

180 185 190

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro

195 200 205

Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu

210 215 220

Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn

225 230 235 240

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

245 250 255

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

260 265 270

Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

275 280 285

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

290 295 300

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

305 310 315 320

Ser Leu Ser Pro

<210> 193

<211> 326

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 193

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Thr Val Glu Arg Lys Ser Cys Val Glu Cys Pro Pro Cys Pro Ala Pro

100 105 110

Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

115 120 125

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

130 135 140

Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly

145 150 155 160

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn

165 170 175

Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp

180 185 190

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro

195 200 205

Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu

210 215 220

Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn

225 230 235 240

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

245 250 255

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

260 265 270

Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

275 280 285

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

290 295 300

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

305 310 315 320

Ser Leu Ser Pro Gly Lys

325

<210> 194

<211> 324

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 194

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Thr Val Glu Arg Lys Ser Cys Val Glu Cys Pro Pro Cys Pro Ala Pro

100 105 110

Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

115 120 125

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

130 135 140

Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly

145 150 155 160

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn

165 170 175

Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp

180 185 190

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro

195 200 205

Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu

210 215 220

Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn

225 230 235 240

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

245 250 255

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

260 265 270

Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

275 280 285

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

290 295 300

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

305 310 315 320

Ser Leu Ser Pro

<210> 195

<211> 115

<212> PRT

<213> Artificial sequence

<220>

<223> Humanized Antibody H Chain

<400> 195

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe

50 55 60

Lys Gly Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser

115

<210> 196

<211> 115

<212> PRT

<213> Artificial sequence

<220>

<223> Humanized Antibody H Chain

<400> 196

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Glu Met His Trp Ile Arg Gln Pro Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Ala Ile Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe

50 55 60

Lys Gly Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser

115

<210> 197

<211> 115

<212> PRT

<213> Artificial sequence

<220>

<223> Humanized Antibody H Chain

<400> 197

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Thr Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Glu Met His Trp Val Arg Gln Ala Pro Gly Glu Gly Leu Glu Trp Met

35 40 45

Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Ser Phe

50 55 60

Gln Asp Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser

115

<210> 198

<211> 115

<212> PRT

<213> Artificial sequence

<220>

<223> Humanized Antibody H Chain

<400> 198

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Glu Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Ala Leu Asn Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe

50 55 60

Lys Gly Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Arg Gly Thr Leu Val Thr

100 105 110

Val Ser Ser

115

<210> 199

<211> 115

<212> PRT

<213> Artificial sequence

<220>

<223> Humanized Antibody H Chain

<400> 199

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Thr Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Glu Met His Trp Ile Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp Ile

35 40 45

Gly Ala Ile Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Ser Phe

50 55 60

Gln Asp Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser

115

<210> 200

<211> 115

<212> PRT

<213> Artificial sequence

<220>

<223> Humanized Antibody H Chain

<400> 200

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Glu Met His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Ala Ile Asn Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe

50 55 60

Lys Gly Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Arg Gly Thr Leu Val Thr

100 105 110

Val Ser Ser

115

<210> 201

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<223> Humanized Antibody L Chain

<400> 201

Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser

20 25 30

Asn Arg Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Asn

85 90 95

Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 202

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<223> Humanized Antibody L Chain

<400> 202

Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser

20 25 30

Asn Arg Asn Thr Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala

35 40 45

Pro Arg Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Asn

85 90 95

Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 203

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<223> Humanized Antibody L Chain

<400> 203

Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ala Ser Glu Ser Leu Val His Ser

20 25 30

Asn Arg Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile

65 70 75 80

Ser Ser Leu Gln Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Asn

85 90 95

Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 204

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<223> Humanized Antibody L Chain

<400> 204

Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Arg Ala Ser Arg Ser Leu Val His Ser

20 25 30

Asn Arg Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Asn

85 90 95

Thr His Val Pro Pro Thr Phe Gly Arg Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 205

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<223> Humanized Antibody L Chain

<400> 205

Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ala Ser Glu Ser Leu Val His Ser

20 25 30

Asn Arg Asn Thr Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala

35 40 45

Pro Arg Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile

65 70 75 80

Ser Ser Leu Gln Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Asn

85 90 95

Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 206

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<223> Humanized Antibody L Chain

<400> 206

Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Arg Ala Ser Arg Ser Leu Val His Ser

20 25 30

Asn Arg Asn Thr Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala

35 40 45

Pro Arg Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Asn

85 90 95

Thr His Val Pro Pro Thr Phe Gly Arg Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 207

<211> 545

<212> PRT

<213> Homo sapiens

<400> 207

Gln Pro Pro Pro Pro Pro Pro Asp Ala Thr Cys His Gln Val Arg Ser

1 5 10 15

Phe Phe Gln Arg Leu Gln Pro Gly Leu Lys Trp Val Pro Glu Thr Pro

20 25 30

Val Pro Gly Ser Asp Leu Gln Val Cys Leu Pro Lys Gly Pro Thr Cys

35 40 45

Cys Ser Arg Lys Met Glu Glu Lys Tyr Gln Leu Thr Ala Arg Leu Asn

50 55 60

Met Glu Gln Leu Leu Gln Ser Ala Ser Met Glu Leu Lys Phe Leu Ile

65 70 75 80

Ile Gln Asn Ala Ala Val Phe Gln Glu Ala Phe Glu Ile Val Val Arg

85 90 95

His Ala Lys Asn Tyr Thr Asn Ala Met Phe Lys Asn Asn Tyr Pro Ser

100 105 110

Leu Thr Pro Gln Ala Phe Glu Phe Val Gly Glu Phe Phe Thr Asp Val

115 120 125

Ser Leu Tyr Ile Leu Gly Ser Asp Ile Asn Val Asp Asp Met Val Asn

130 135 140

Glu Leu Phe Asp Ser Leu Phe Pro Val Ile Tyr Thr Gln Leu Met Asn

145 150 155 160

Pro Gly Leu Pro Asp Ser Ala Leu Asp Ile Asn Glu Cys Leu Arg Gly

165 170 175

Ala Arg Arg Asp Leu Lys Val Phe Gly Asn Phe Pro Lys Leu Ile Met

180 185 190

Thr Gln Val Ser Lys Ser Leu Gln Val Thr Arg Ile Phe Leu Gln Ala

195 200 205

Leu Asn Leu Gly Ile Glu Val Ile Asn Thr Thr Asp His Leu Lys Phe

210 215 220

Ser Lys Asp Cys Gly Arg Met Leu Thr Arg Met Trp Tyr Cys Ser Tyr

225 230 235 240

Cys Gln Gly Leu Met Met Val Lys Pro Cys Gly Gly Tyr Cys Asn Val

245 250 255

Val Met Gln Gly Cys Met Ala Gly Val Val Glu Ile Asp Lys Tyr Trp

260 265 270

Arg Glu Tyr Ile Leu Ser Leu Glu Glu Leu Val Asn Gly Met Tyr Arg

275 280 285

Ile Tyr Asp Met Glu Asn Val Leu Leu Gly Leu Phe Ser Thr Ile His

290 295 300

Asp Ser Ile Gln Tyr Val Gln Lys Asn Ala Gly Lys Leu Thr Thr Thr

305 310 315 320

Ile Gly Lys Leu Cys Ala His Ser Gln Gln Arg Gln Tyr Arg Ser Ala

325 330 335

Tyr Tyr Pro Glu Asp Leu Phe Ile Asp Lys Lys Val Leu Lys Val Ala

340 345 350

His Val Glu His Glu Glu Thr Leu Ser Ser Arg Arg Arg Glu Leu Ile

355 360 365

Gln Lys Leu Lys Ser Phe Ile Ser Phe Tyr Ser Ala Leu Pro Gly Tyr

370 375 380

Ile Cys Ser His Ser Pro Val Ala Glu Asn Asp Thr Leu Cys Trp Asn

385 390 395 400

Gly Gln Glu Leu Val Glu Arg Tyr Ser Gln Lys Ala Ala Arg Asn Gly

405 410 415

Met Lys Asn Gln Phe Asn Leu His Glu Leu Lys Met Lys Gly Pro Glu

420 425 430

Pro Val Val Ser Gln Ile Ile Asp Lys Leu Lys His Ile Asn Gln Leu

435 440 445

Leu Arg Thr Met Ser Met Pro Lys Gly Arg Val Leu Asp Lys Asn Leu

450 455 460

Asp Glu Glu Gly Phe Glu Ala Gly Asp Cys Gly Asp Asp Glu Asp Glu

465 470 475 480

Cys Ile Gly Gly Ala Gly Asp Gly Met Ile Lys Val Lys Asn Gln Leu

485 490 495

Arg Phe Leu Ala Glu Leu Ala Tyr Asp Leu Asp Val Asp Asp Ala Pro

500 505 510

Gly Asn Ser Gln Gln Ala Thr Pro Lys Asp Asn Glu Ile Ser Thr Phe

515 520 525

His Asn Leu Gly Asn Val His Ser Pro Leu Lys His His His His

530 535 540

His

545

<210> 208

<211> 32

<212> DNA

<213> Artificial sequence

<220>

<223> Artificially synthesized primer sequence

<400> 208

ggatcctgcg catgaaaaag cctgaactca cc 32

<210> 209

<211> 29

<212> DNA

<213> Artificial sequence

<220>

<223> Artificially synthesized primer sequence

<400> 209

gcggccgcct attcctttgc cctcggacg 29

<210> 210

<211> 10939

<212> DNA

<213> Cricetulus griseus

<400> 210

gagctcaatt aaccctcact aaagggagtc gactcgatcc tttacagaaa acttgcaaac 60

cctcttggag tagaaaagta gtagtatctg acacaagtat cagcaaaatg caaacttctc 120

cccatcccca gaaaaccatt ataaaaaccc ccatatctta tgcccaactg tagtgatata 180

ttatttatga tttattaaaa cttgcttaag gattcagaaa gcaaagtcag ccttaagcta 240

tagagaccag gcagtcagtg gtggtacaca cctttaatcc caggactcag gattaagaag 300

tagacggacc tctgttagtt caagtctacc attacctaca caagagtgaa gagtaaccga 360

tctcatgcctttgatcccag cagctgggat catgtgcatt caatcccagc attcggggagt 420

tatataagac aggagcaagg tctcagagct ggcattcatt ctccagccac attgaggata 480

ggaaaacatt gaagtgtcag gatgctgagg agaggcagca gtttgaggtt tggtagaacc 540

aggatcaccttttggtctga ggtagagtaa gaactgtggc tggctgcttt gcttttctga 600

tcttcagctt gaagcttgaa ctccaatatt tgtctctggg tctattatta tcatgttaca 660

cctaacttta aagctgattt acgcaagaca gttgtaggtg gacctttctt tcctgcccac 720

cagttcccaa ataactgaca cggagactca atattaatta taaatgattg gttaatagct 780

cagtcttgtt actggctaac tcttacattt taaattaact catttccatc cctttacttg 840

ctgccatgtg gttcatggct tgttcaagtc ctgcttcttc tgtctctggc tggtgatgcc 900

tctggttctg ccctttatcc cagaattctc ctagtctggc tctcctgccc agctataggc 960

cagtcagctg tttattaacc aatgagaata atacatattt atagtgtaca aagattgctc 1020

ctcaacaccc aattttttat gtgcaacctg agaatctgga ctcattgccc tcatgcttgc 1080

agaggcggca cccttaccca ctaagccacc tttctagccc tgttgctttt gttttttgag 1140

acaggttcca ctatgtagcc caggctggcc tcaaactgac cattctcctg cctaaacctc 1200

ccgaacactg gaattatagt caaggcctac ctgccctggc attttcacac ttttatttcc 1260

tggctgagtc cattgacttt acactcatca aggttgaacc agttggagtt taattacagt 1320

gccaatcgca ctgaatccca cataatcaaa caacttcaag gaagcaaaaa accagttttt 1380

cctgaagatc aatgtcagct tgcctgattc agaatagacc cccgaaaaaa ggcaaatgct 1440

tgataaccaa tttcttctta ttgttcaatc ccctgctgct gtgtgtaagc tcctgagaaa 1500

ggacagtaag gggacattca tgatcagaga aagagcccca actccccccc cagccccacc 1560

cccaccctgt ccacagtctg ttggtttggt ttccccctgg ctgacaccca gaaatcacaa 1620

cataatcacc taggtcactg taacaagttc ctttctggaa aatgctacaa atgatattgg 1680

taacatgagt aatgaataat gcctggagtc caactccctt gtgacccagc aatgttttcc 1740

gtgggtgctc ccttccccag ctgcaggcct gacatgtacc ttaaaaagcc tcccctggag 1800

gacagaattt tgtgggtact atagtgttct cacaaatact tcccctaata cccttactta 1860

gttaccataa ataacatgca gcccctggtg aggcacacag ggctccaatg tacagcttct 1920

cagacactgc aggaaccttc ctctcctaat gcagcactgg tctcttcagg ctggacagca 1980

ggaacccata ccactccaat cctagtgtgg agtagagctg tctacgaaaa ccagcagatc 2040

tatagctaaa tgtgtttcaa ttttatgctt tgacaaattg tactgacccc accccccaccc 2100

cttccccctt gctgtgctgg gaattgaacc caggaccttg tgcatgccag gcaagtactc 2160

taacactgag ctatagcccc aatctttcat ccaagtctct atgtgtgccc acactcgctt 2220

tttattttga gacaaaaggt tcttattttg agataaggtc tcactatgtt gccttgactt 2280

ttttttttttttttttttga acttttgacc ttcctacctc agctgagact acaagtcttt 2340

taccatcagg cccggctgat ggtaaaataa cagtatttga aatagtttaa acacatcatc 2400

ttaatggtca accacacaat ttccgaaatg ttgctggctc agtctggggc aaacctgtcc 2460

gccccaacat tggtgctagg aagaaagcac agacaagtag ccctcccagc tcaggagtaa 2520

aagacctgga gggggtggcc cacttcggtc aagttcacgg gatggggagg ggtaccctcc 2580

tccagtagtg gtggtatttg gcagttcctc caccgacgcc ctctggaagc acctgcttgg 2640

acccgcaaag ccaggaatgc agcttcctca agggactcgc cagcgagggt aacaggacag 2700

aggcgtccca agagggctgg ggcggaaggg ggaagacagg gtcggcctta gatagggcaa 2760

agggccttct ggctgtgttc ccggggtaac cgccccacca cgcctggagc ccgacgtggc 2820

gagcgatggg gacagcgagc aggaagtcgt actggggagg gccgcgtagc agatgcagcc 2880

gagggcggcg ctgccaggta cacccgaggg caccgcgggg gtgagcgcca ggtccctgaa 2940

ccagccaggc ctccagagcc gagtccggcg gaccgacggt acgttctgga atgggaaggg 3000

atccgggaca ccgaattgct gcattgaggg gctcagaggt tctgatgtgg gagtccagaa 3060

agggttttat ctaccggagg tgatgtgact tccggcctct ggaagtgctg ttggagtctc 3120

tgggaccttg ggtcctctcg actaggtttg gaaggggtga aataggggta gggagaaagg 3180

agaggactgc agcaatgtct tcccgaacga cctgggttcg ggaggggtcg aaggacaagg 3240

ggctgttgtg gggggtcttc agacgcggag gggtggtatt ctattttctg ggaagatggt 3300

gtcgatgcac ttgaccaagt ctagtcgatc tgaagaggct aggggaacag acagtgagag 3360

aggatggtgg agggagtggc agaacccttc cagaaactgg gagaggctct agcacctgca 3420

accccttccc tggcctccgg ggagtcccag aagagggcag gaccatggac acaggtgcat 3480

tcgtgccggc gcgctccggc ctggcgaagg tgcgcgctct tggaggccgc gggagggcca 3540

gacgcgcgcc cggagagctg gccctttaag gctacccgga ggcgtgtcag gaaatgcgcc 3600

ctgagcccgc ccctcccgga acgcggcccg agacctggca agctgagacg gaactcggaa 3660

ctagcactcg gctcgcggcc tcggtgaggc cttgcgcccg ccatgcctct gtcattgccc 3720

ctcgggccgc ctccctgaac ctccgtgacc gccctgcagt cctccctccc ccccttcgac 3780

tcggcgggcg cttccgggcg ctcccgcagc ccgccctcca cgtagcccac acctccctct 3840

cggcgctccg cttcccacgc ggtccccgac ctgttctttc ctcctccacc ctgcccttct 3900

gtccctctcc cttcctttct cccctcgact cgtccctatt aggcaacagc ccctgtggtc 3960

cagccggcca tggctgtcaa ggctcacacc cttagctagg ccccttctcc cttccctggg 4020

tcttgtctca tgaccccctg ccccgcccgg gagcgagcgc gatgtggagc agtgcctctg 4080

gcaagcagaa cttcacccaa gccatgtgac aattgaaggc tgtaccccca gaccctaaca 4140

tcttggagcc ctgtagacca gggagtgctt ctggccgtgg ggtgacctag ctcttctacc 4200

accatgaaca gggcccctct gaagcggtcc aggatcctgc gcatggcgct gactggaggc 4260

tccactgcct ctgaggaggc agatgaagac agcaggaaca agccgtttct gctgcgggcg 4320

ctgcagatcg cgctggtcgt ctctctctac tgggtcacct ccatctccat ggtattcctc 4380

aacaagtacc tgctggacag cccctccctg cagctggata cccctatctt cgtcactttc 4440

taccaatgcc tggtgacctc tctgctgtgc aagggcctca gcactctggc cacctgctgc 4500

cctggcaccg ttgacttccc caccctgaac ctggacctta aggtggcccg cagcgtgctg 4560

ccactgtcgg tagtcttcat tggcatgata agtttcaata acctctgcct caagtacgta 4620

ggggtggcct tctacaacgt ggggcgctcg ctcaccaccg tgttcaatgt gcttctgtcc 4680

tacctgctgc tcaaacagac cacttccttc tatgccctgc tcacatgtgg catcatcatt 4740

ggtgagtggg gcccgggggc tgtgggagca ggatgggcat cgaactgaag ccctaaaggt 4800

caacactgta ggtaccttta cttactgtcc caggtccctt gcatcagcag ttacaggaag 4860

agccctgtag aaaacaaata acttccttat ggtcattcaa caagttaggg acccagccag 4920

ggtgaaaata atgttagcag caactacagc aaagatggct ctcgccactt gcatgattaa 4980

aatgtgccag gtactcagat ctaagcattg gatccacatt aactcaacta atccctatta 5040

caaggtaaaa tatatccgaa ttttacagag ggaaaaccaa ggcacagaga ggctaagtag 5100

cttgaccagg atcacacagc taataatcac tgacatagct gggatttaaa cataagcagt 5160

tacctccata gatcacacta tgaccaccat gccactgttc cttctcaaga gttccaggat 5220

cctgtctgtc cagttctctt taaagaggac aacacatctg acattgctac cttgaggtaa 5280

catttgaaat agtgggtaga catatgtttt aagttttat cttacttttt atgtgtgtgt 5340

gtttgggggg ccaccacagt gtatgggtgg agataagggg acaacttaag aattggtcct 5400

ttctcccacc acatgggtgc tgaggtctga actcaggtca tcaggattgg cacaaatccc 5460

tttacccact gagccatttc actggtccaa tatatgtgtg cttttaagag gctttaacta 5520

ttttcccaga tgtgaatgtc ctgctgatca ttatcccctt ttacccggaa gccctctggg 5580

aggtgccatc cctgtggtcg tctgcataca aatggggaaa ctgcaactca gagaaacaag 5640

gctacttgcc agggccccac aagtaagata ggctggggatg ccatcccaga ctggccacac 5700

tccctggcct gtgcttcaag ccagtttact ttgttcctgc ccattggaag ttagcatgtt 5760

gcagtcaaac acaataacta caggccaaaa gtgcttttaa attaaagtca gatgaacttt 5820

taaacatcca gagctcctca actgcaggag ttacaacctg attctgcaac catctttgca 5880

gtgcccggta gtcatatgta gctagaggct cttggctagg acagcatgtg ttaggaaaca 5940

tctggccctg agatcattga attgagtgac tgctgggtga caaagaccaa ggcatccgtt 6000

ccctgagagt cctgggcaag cagcaatgtg accttcattt gtacctactc aggttcttta 6060

tctgtcctgt ttgacctact tagtctcctc tggtgtctca gaggcccagg ctgggtactc 6120

tggatgtcag gatcaggcca atgcgcacat ctgccctaga aatgtccccc tggttgagca 6180

gctcctgaat ccatcggtaa agggtctgga ccagggagga gtcagataaa aagctgacag 6240

cactggggga ctccatgggg aactcccacc tgcccccaca catccatcct aagagaactg 6300

gtattccttg tttcctcttt gtcctacaag gcaccctggg atcccacttc agtctcccag 6360

ccttgccagg gttagagggc atgagcctcc ttgtggggaa tttagatgca agaaggtaca 6420

gtcactagag aacctgagct cagatcccca aagtaaccag tacctgatag tgaggcagct 6480

gagaaccgca gcagcctgcc tgagtggctg aactctgcgg cctccggaac tggccccaac 6540

tgttgggtct cctcttcctt cctcctgtga gggagggccc atctctgata agtgctgtgg 6600

ggactctaga gtagggagga ggaggagcaa tctaagcagg ccttactgag aagtccttgc 6660

tggcatgtgg ctgcctgagg agtacagact gggaacaccc atttgaatga gtaaggtttt 6720

tcctgaaggc catggggagc cacggaggaa aatcatttta gttacaagac aaagagtaga 6780

ttggttaaca tgggagcaag gacatggccc caattttcat agatgaagga aattggaact 6840

cagagaggtt aagtaacttc tcccaaatag ctcagcttca aaatcacaga acagtcagag 6900

tctagatctc tctgatgcct gtgatggtcc tgccattcca tgttgctgat ccctgtggca 6960

tcagtaagcc tctaccttgt gggaatgcag gatctaaatg aagagaggaa gtgctggccc 7020

catgctgtgg tctggaaagc tatgcaggct ctttgagcag agagtgaccc acaagtgaat 7080

agagtcctat gagactcaaa gcaacatcca cccttaagca gctctaacca aatgctcaca 7140

ctgagggagc caaagccaag ttagagtcct gtgcttgccc aaggtcactt tgcctggccc 7200

tcctcctata gcacccgtgt tatcttatag ccctcattac agtgattaca attataatta 7260

gagaggtaac agggccacac tgtccttaca cattcccctg ctagattgta gctgggagag 7320

ggggagatgt aggtggctgg gggagtggga gggaagatgc agattttcat tctgggctct 7380

actccctcag ccattttttg gtgtgggagt tagactttgg atatgttgat gatgaggtaa 7440

gggccacaga acagtctgaa ctgtggtatc agaatcctgt ccctctccct ctctcctcat 7500

ccctcttcac cttgtcactc ctctgtctgc tacaggtggt ttctggctgg gtatagacca 7560

agaggggagct gagggcaccc tgtccctcat aggcaccatc ttcggggtgc tggccagcct 7620

ctgcgtctcc ctcaatgcca tctatacccaa gaaggtgctc ccagcagtgg acaacagcat 7680

ctggcgccta accttctata acaatgtcaa tgcctgtgtg ctcttcttgc ccctgatggt 7740

tctgctgggt gagctccgtg ccctccttga ctttgctcat ctgtacagtg cccacttctg 7800

gctcatgatg acgctgggtg gcctcttcgg ctttgccatt ggctatgtga caggactgca 7860

gatcaaattc accagtcccc tgacccacaa tgtatcaggc acagccaagg cctgtgcgca 7920

gacagtgctg gccgtgctct actatgaaga gactaagagc ttcctgtggt ggacaagcaa 7980

cctgatggtg ctgggtggct cctcagccta tacctgggtc aggggctggg agatgcagaa 8040

gacccaagag gaccccagct ccaaagaggg tgagaagagt gctattgggg tgtgagcttc 8100

ttcagggacc tgggactgaa cccaagtggg gcctacacag cactgaaggc ttcccatgga 8160

gctagccagt gtggccctga gcaatactgt ttacatcctc cttggaatat gatctaagag 8220

gagccagggt ctttcctggt aatgtcagaa agctgccaaa tctcctgtct gccccatctt 8280

gttttgggaa aaccctacca ggaatggcac ccctacctgc ctcctcctag agcctgtcta 8340

cctccatatc atctctgggg ttgggaccag ctgcagcctt aaggggctgg attgatgaag 8400

tgatgtcttc tacacaaggg agatgggttg tgatcccact aattgaaggg atttgggtga 8460

ccccacacct ctgggatcca gggcaggtag agtagtagct taggtgctat taacatcagg 8520

aacacctcag cctgcctttg aagggaagtg ggagcttggc caagggagga aatggccatt 8580

ctgccctctt cagtgtggat gagtatggca gacctgttca tggcagctgc accctggggt 8640

ggctgataag aaaacattca cctctgcatt tcatatttgc agctctagaa cggggggagag 8700

ccacacatct tttacgggtt aagtagggtg atgagctcct ccgcagtccc taaccccagc 8760

tttacctgcc tggcttccct tggcccagct acctagctgt actccctttc tgtactcttc 8820

tcttctccgt catggcctcc cccaacacct ccatctgcag gcaggaagtg gagtccactt 8880

gtaacctctg ttcccatgac agagcccttt gaatacctga acccctcatg acagtaagag 8940

acatttatgt tctctggggc tggggctgaa ggagcccact ggttctcact tagcctatct 9000

ggctcctgtc acaaaaaaaa aaaaagaaaa aaaaaaagca taaaccaagt tactaagaac 9060

agaagttggt ttataacgtt ctggggcagc aaagcccaga tgaagggacc catcgaccct 9120

ctctgtccat atcctcatgc tgcagaagta caggcaagct cctttaagcc tcatatagga 9180

acactagcct cactcatgag ggttttactc catgacctgt caacctcaaa gccttcaaca 9240

tgaggactcc aacgtaaatt tggggacaga agcactcaga ccataccccca gcaccacacc 9300

ctcctaacct cagggtagct gtcattctcc tagtctcctc tcttgggcct ttagaacccc 9360

catttccttg gggtaatgtc tgatgttttt gtccctgtca taaaaagatg gagagactgt 9420

gtccagcctt tgattcctac ttcctacaat cccaggttct aatgaagttt gtggggcctg 9480

atgccctgag ttgtatgtga tttaataata aaaaagcaag atacagcatg tgtgtggact 9540

gagtgagggc cacagggatc taaaagccaa gtgtgagggg acccagctac agcaggcagc 9600

atcctgagcc tggaatctct tcaggacaag aattctccat atacctacct actctgggga 9660

gtaggtggcc agagttcaag cttcccttag taccaactac cactggctgt gctcttactg 9720

aaggcagaca tggcactgag tgctgtccat ctgtcactca tctccacagc cattcctaat 9780

gtgtggggtg ggagccatca ccaaacccca ttttcagata aggacacagg ctcagagagg 9840

cttgtgtgga gaaaagtagc agcagaattc agagagctgg gtctcctgca gcaccttgga 9900

ctgccagcag ccacagtgct tgtcacacag cacatactca aaagaatgcc agccccctca 9960

gcctagagtg cctggccttt ctttcagatg aggaagaggg tcaaagctgt tagcttgccc 10020

accatatgac cacatacatg accaacagct tgagggaggg aggattactg tggctcccag 10080

cctgagaggt gggacaccca aatgtattag gtccttgaat cagggctgac cttgtgattc 10140

agtcactcct accagaatgc tggggaatgg ggatgccaaa ggcaaaggag gctttctaag 10200

gtgtggtgta agataggcat ttctgcttcc atgtacacct gtgagcagag taggaaggcc 10260

ctgtggagaa tatatcccac aaaccagtag cccttcctgg cagtgggtga atactgccac 10320

cctatacccc tatgcaaggc cagtagaacc acccaaccca caacatctag agaaattaca 10380

ggtcatctta agcctctaaa ttgtggagaa actcgacatg cgcacgattc ctaacctgct 10440

agcctagggt gcggggtgga taatttaagg aaactggggt ttctttataga atcggaggct 10500

ccatgaagtc accctgacaa gaggtcagca atagccagca gcagtggcta ctcctaagcc 10560

tccagacaga gcaccctgtg aatgtacctt attctcacat ctgggtgtct ataggtgtga 10620

ctgggtcaga tgtcacccag gccattgcaa tgggccctta gccccatggg gtgttggggat 10680

agcagccaag cagctcccat gctgagatac tgcctgcagt agactgatgg ataagaaaac 10740

aaggcccaaa atgttttctt tccagacttg atctttcttt gttcaaaaat gctgttttcc 10800

cttaaacttg cccaaaccca ttgttttgca gttgaggaaa ataaggcata gaaagattaa 10860

aggaagtttc tgaggttaca gagcaaagta ctggcttcac ctgaaataga caggtgtgcc 10920

ctgatcctga tttgagctc 10939

<210> 211

<211> 33

<212> DNA

<213> Artificial sequence

<220>

<223> Artificially synthesized primer sequence

<400> 211

atgcatgcca ccatgaaaaa gcctgaactc acc 33

<210> 212

<211> 28

<212> DNA

<213> Artificial sequence

<220>

<223> Artificially synthesized primer sequence

<400> 212

ggatcccagg ctttacactt tatgcttc 28

<210> 213

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> Artificially synthesized primer sequence

<400> 213

gctgtctgga gtactgtgca tctgc 25

<210> 214

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223> Artificially synthesized primer sequence

<400> 214

ggaatgcagc ttcctcaagg gactcgc 27

<210> 215

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223> Artificially synthesized primer sequence

<400> 215

tgcatcaggt cggagacgct gtcgaac 27

<210> 216

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223> Artificially synthesized primer sequence

<400> 216

gcactcgtcc gagggcaaag gaatagc 27

<210> 217

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Artificially synthesized primer sequence

<400> 217

tgtgctggga attgaaccca ggac 24

<210> 218

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> Artificially synthesized primer sequence

<400> 218

ctacttgtct gtgctttctt cc 22

<210> 219

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223> Artificially synthesized primer sequence

<400> 219

ctcgactcgt ccctattagg caacagc 27

<210> 220

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223> Artificially synthesized primer sequence

<400> 220

tcagaggcag tggagcctcc agtcagc 27

<210> 221

<211> 449

<212> PRT

<213> Homo sapiens

<400> 221

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp

20 25 30

His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp

35 40 45

Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Val Leu Ala Arg Ile Thr Ala Met Asp Tyr Trp Gly Gln Gly

100 105 110

Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

Lys

<210> 222

<211> 449

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 222

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Asp Asp

20 25 30

His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp

35 40 45

Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu

50 55 60

Lys Gly Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Ala Tyr Tyr Cys

85 90 95

Ala Arg Val Leu Ala Arg Ile Thr Ala Met Asp Tyr Trp Gly Glu Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

Lys

<210> 223

<211> 449

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 223

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Asp Asp

20 25 30

His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp

35 40 45

Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu

50 55 60

Gln Asp Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Leu Leu Ala Arg Ala Thr Ala Met Asp Tyr Trp Gly Gln Gly

100 105 110

Ile Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

Lys

<210> 224

<211> 214

<212> PRT

<213> Homo sapiens

<400> 224

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Gly Gln Gly Asn Arg Leu Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 225

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 225

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Ser Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Glu Leu Leu Ile

35 40 45

Tyr Tyr Gly Ser Glu Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Glu Ala

65 70 75 80

Glu Asp Ala Ala Thr Tyr Tyr Cys Gly Gln Gly Asn Arg Leu Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Glu Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 226

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 226

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Ser Val Thr Ile Thr Cys Gln Ala Ser Glu Asp Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Glu Leu Leu Ile

35 40 45

Tyr Tyr Gly Ser Glu Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Glu Ala

65 70 75 80

Glu Asp Ala Ala Thr Tyr Tyr Cys Gly Gln Gly Asn Arg Leu Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Glu Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 227

<211> 449

<212> PRT

<213> Homo sapiens

<400> 227

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp

20 25 30

His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp

35 40 45

Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gln Gly

100 105 110

Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

Lys

<210> 228

<211> 449

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 228

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Asp Asp

20 25 30

His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp

35 40 45

Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu

50 55 60

Lys Gly Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Ala Tyr Tyr Cys

85 90 95

Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

Lys

<210> 229

<211> 214

<212> PRT

<213> Homo sapiens

<400> 229

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 230

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 230

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Ser Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Glu Leu Leu Ile

35 40 45

Tyr Tyr Gly Ser Glu Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Glu Ala

65 70 75 80

Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Ser Leu Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Glu Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 231

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 231

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Ser Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Glu Leu Leu Ile

35 40 45

Tyr Tyr Gly Ser Glu Glu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Glu Ala

65 70 75 80

Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Ser Leu Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Glu Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 232

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 232

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Ser Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Glu Leu Leu Ile

35 40 45

Tyr Tyr Gly Ser Glu Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Glu Ala

65 70 75 80

Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Ser Leu Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Glu Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 233

<211> 445

<212> PRT

<213> Homo sapiens

<400> 233

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Glu Met His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Ala Ile Asn Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe

50 55 60

Lys Gly Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Arg Gly Thr Leu Val Thr

100 105 110

Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro

115 120 125

Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val

130 135 140

Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala

145 150 155 160

Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly

165 170 175

Leu Tyr Ser Leu Ser Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly

180 185 190

Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys

195 200 205

Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys

210 215 220

Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu

225 230 235 240

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

245 250 255

Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys

260 265 270

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

275 280 285

Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu

290 295 300

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

305 310 315 320

Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys

325 330 335

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

340 345 350

Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

355 360 365

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

370 375 380

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

385 390 395 400

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

405 410 415

Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

420 425 430

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 234

<211> 445

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 234

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe

50 55 60

Lys Gly Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro

115 120 125

Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val

130 135 140

Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala

145 150 155 160

Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly

165 170 175

Leu Tyr Ser Leu Ser Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly

180 185 190

Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys

195 200 205

Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys

210 215 220

Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu

225 230 235 240

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

245 250 255

Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys

260 265 270

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

275 280 285

Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu

290 295 300

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

305 310 315 320

Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys

325 330 335

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

340 345 350

Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

355 360 365

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

370 375 380

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

385 390 395 400

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

405 410 415

Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

420 425 430

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 235

<211> 445

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 235

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Thr Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Glu Met His Trp Ile Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp Ile

35 40 45

Gly Ala Ile Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Glu Ser Phe

50 55 60

Gln Asp Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro

115 120 125

Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val

130 135 140

Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala

145 150 155 160

Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly

165 170 175

Leu Tyr Ser Leu Ser Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly

180 185 190

Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys

195 200 205

Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys

210 215 220

Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu

225 230 235 240

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

245 250 255

Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys

260 265 270

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

275 280 285

Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu

290 295 300

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

305 310 315 320

Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys

325 330 335

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

340 345 350

Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

355 360 365

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

370 375 380

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

385 390 395 400

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

405 410 415

Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

420 425 430

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 236

<211> 219

<212> PRT

<213> Homo sapiens

<400> 236

Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Arg Ala Ser Arg Ser Leu Val His Ser

20 25 30

Asn Arg Asn Thr Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala

35 40 45

Pro Arg Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Asn

85 90 95

Thr His Val Pro Pro Thr Phe Gly Arg Gly Thr Lys Leu Glu Ile Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 237

<211> 219

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 237

Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser

20 25 30

Asn Arg Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Asn

85 90 95

Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 238

<211> 219

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 238

Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Gln Ala Ser Glu Ser Leu Val His Ser

20 25 30

Asn Arg Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Asn

85 90 95

Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Glu

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 239

<211> 447

<212> PRT

<213> Homo sapiens

<400> 239

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr

20 25 30

Ile Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Leu Ile Asn Pro Tyr Asn Gly Gly Thr Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Gly Tyr Asp Asp Gly Pro Tyr Thr Met Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Glu Ser Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Ser Cys

210 215 220

Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

260 265 270

Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser

290 295 300

Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile

325 330 335

Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 240

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 240

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr

20 25 30

Ile Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Leu Ile Asn Pro Tyr Asn Gly Gly Thr Ser Tyr Asn Gln Gln Phe

50 55 60

Gln Asp Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Gly Tyr Asp Asp Gly Pro Tyr Thr Met Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Glu Ser Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Ser Cys

210 215 220

Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

260 265 270

Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser

290 295 300

Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile

325 330 335

Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 241

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 241

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr

20 25 30

Ile Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Leu Ile Asn Pro Tyr Asn Gly Gly Thr Ser Tyr Asn Asp Gln Phe

50 55 60

Gln Asp Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Gly Tyr Asp Asp Gly Pro Tyr Thr Met Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Glu Ser Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Ser Cys

210 215 220

Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

260 265 270

Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser

290 295 300

Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile

325 330 335

Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 242

<211> 214

<212> PRT

<213> Homo sapiens

<400> 242

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Thr Ser Glu Asn Ile Tyr Ser Phe

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Asn Ala Lys Thr Leu Ala Lys Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His His Tyr Glu Ser Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 243

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 243

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Gln Thr Ser Glu Asp Ile Tyr Ser Phe

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Asn Ala Gln Thr Glu Ala Gln Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His His Tyr Glu Ser Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 244

<211> 445

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 244

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr

20 25 30

Ile Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Leu Ile Asn Pro Tyr Asn Gly Gly Thr Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Gly Tyr Asp Asp Gly Pro Tyr Thr Met Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Ser Cys

210 215 220

Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu

260 265 270

Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser

290 295 300

Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile

325 330 335

Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

<210> 245

<211> 445

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized peptide sequence

<400> 245

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr

20 25 30

Ile Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Leu Ile Asn Pro Tyr Asn Gly Gly Thr Ser Tyr Asn Gln Gln Phe

50 55 60

Gln Asp Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Gly Tyr Asp Asp Gly Pro Tyr Thr Met Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Ser Cys

210 215 220

Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu

260 265 270

Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser

290 295 300

Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile

325 330 335

Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

<210> 246

<211> 514

<212> PRT

<213> Homo sapiens

<400> 246

Met Met Trp Thr Trp Ala Leu Trp Met Leu Pro Ser Leu Cys Lys Phe

1 5 10 15

Ser Leu Ala Ala Leu Pro Ala Lys Pro Glu Asn Ile Ser Cys Val Tyr

20 25 30

Tyr Tyr Arg Lys Asn Leu Thr Cys Thr Trp Ser Pro Gly Lys Glu Thr

35 40 45

Ser Tyr Thr Gln Tyr Thr Val Lys Arg Thr Tyr Ala Phe Gly Glu Lys

50 55 60

His Asp Asn Cys Thr Thr Asn Ser Ser Thr Ser Glu Asn Arg Ala Ser

65 70 75 80

Cys Ser Phe Phe Leu Pro Arg Ile Thr Ile Pro Asp Asn Tyr Thr Ile

85 90 95

Glu Val Glu Ala Glu Asn Gly Asp Gly Val Ile Lys Ser His Met Thr

100 105 110

Tyr Trp Arg Leu Glu Asn Ile Ala Lys Thr Glu Pro Pro Lys Ile Phe

115 120 125

Arg Val Lys Pro Val Leu Gly Ile Lys Arg Met Ile Gln Ile Glu Trp

130 135 140

Ile Lys Pro Glu Leu Ala Pro Val Ser Ser Asp Leu Lys Tyr Thr Leu

145 150 155 160

Arg Phe Arg Thr Val Asn Ser Thr Ser Trp Met Glu Val Asn Phe Ala

165 170 175

Lys Asn Arg Lys Asp Lys Asn Gln Thr Tyr Asn Leu Thr Gly Leu Gln

180 185 190

Pro Phe Thr Glu Tyr Val Ile Ala Leu Arg Cys Ala Val Lys Glu Ser

195 200 205

Lys Phe Trp Ser Asp Trp Ser Gln Glu Lys Met Gly Met Thr Glu Glu

210 215 220

Glu Ala Pro Cys Gly Leu Glu Leu Trp Arg Val Leu Lys Pro Ala Glu

225 230 235 240

Ala Asp Gly Arg Arg Pro Val Arg Leu Leu Trp Lys Lys Ala Arg Gly

245 250 255

Ala Pro Val Leu Glu Lys Thr Leu Gly Tyr Asn Ile Trp Tyr Tyr Pro

260 265 270

Glu Ser Asn Thr Asn Leu Thr Glu Thr Met Asn Thr Thr Asn Gln Gln

275 280 285

Leu Glu Leu His Leu Gly Gly Glu Ser Phe Trp Val Ser Met Ile Ser

290 295 300

Tyr Asn Ser Leu Gly Lys Ser Pro Val Ala Thr Leu Arg Ile Pro Ala

305 310 315 320

Ile Gln Glu Lys Ser Phe Gln Cys Ile Glu Val Met Gln Ala Cys Val

325 330 335

Ala Glu Asp Gln Leu Val Val Lys Trp Gln Ser Ser Ala Leu Asp Val

340 345 350

Asn Thr Trp Met Ile Glu Trp Phe Pro Asp Val Asp Ser Glu Pro Thr

355 360 365

Thr Leu Ser Trp Glu Ser Val Ser Gln Ala Thr Asn Trp Thr Ile Gln

370 375 380

Gln Asp Lys Leu Lys Pro Phe Trp Cys Tyr Asn Ile Ser Val Tyr Pro

385 390 395 400

Met Leu His Asp Lys Val Gly Glu Pro Tyr Ser Ile Gln Ala Tyr Ala

405 410 415

Lys Glu Gly Val Pro Ser Glu Gly Pro Glu Thr Lys Val Glu Asn Ile

420 425 430

Gly Val Lys Thr Val Thr Ile Thr Trp Lys Glu Ile Pro Lys Ser Glu

435 440 445

Arg Lys Gly Ile Ile Cys Asn Tyr Thr Ile Phe Tyr Gln Ala Glu Gly

450 455 460

Gly Lys Gly Phe Ser Lys Thr Val Asn Ser Ser Ile Leu Gln Tyr Gly

465 470 475 480

Leu Glu Ser Leu Lys Arg Lys Thr Ser Tyr Ile Val Gln Val Met Ala

485 490 495

Ser Thr Ser Ala Gly Gly Thr Asn Gly Thr Ser Ile Asn Phe Lys Thr

500 505 510

Leu Ser

Claims (14)

1. A method for controlling the pharmacokinetics of a polypeptide comprising an antibody variable region in plasma, the method comprising altering the isoelectric point of the polypeptide by changing the charge of at least one exposed amino acid residue on the surface of a complementarity-determining region (CDR) of the polypeptide, wherein controlling the pharmacokinetics in plasma refers to prolonging or shortening the half-life of the polypeptide in plasma, wherein a decrease in the isoelectric point of the polypeptide results in a prolonged half-life in plasma, and an increase in the isoelectric point of the polypeptide results in a shortened half-life in plasma, wherein the amino acid residue exposed on the surface of the CDR is at least one amino acid residue selected from amino acid residues at positions 31, 61, 62, 64, and 65 of the Kabat number in the heavy chain variable region and amino acid residues at positions 24, 27, 53, 54, and 55 of the Kabat number in the light chain variable region, wherein the charge of the amino acid residue is altered by amino acid substitution, and the modified amino acid is selected from amino acid residues in either group (a) or (b): (a) Glutamic acid (E) and aspartic acid (D); and (b) Lysine (K), arginine (R), and histidine (H), The polypeptide containing the antibody variable region further includes an IgG Fc region capable of binding FcRn. 2. The method of claim 1, wherein the polypeptide comprising the antibody variable region is an IgG antibody. 3. The method of claim 1, wherein the polypeptide comprising the antibody variable region is a multispecific polypeptide that binds to at least two types of antigens. 4. The method of claim 1, wherein the polypeptide comprising the antibody variable region is a chimeric antibody, a humanized antibody, or a human antibody. 5. The method of claim 1, wherein the polypeptide prior to modification is a humanized antibody. 6. The method of claim 1, wherein the change in the charge of the amino acid residues results in a change in the theoretical isoelectric point of 1.0 or higher. 7. The method of claim 1, wherein pharmacokinetic control refers to increasing or decreasing any one of the following parameters: plasma clearance CL, area under the concentration curve AUC, mean plasma residence time, and plasma half-life t1/2. 8. A method for manufacturing a polypeptide, said polypeptide comprising an antibody variable region whose pharmacokinetics in plasma are controlled, the method comprising: altering the isoelectric point of the polypeptide comprising the antibody variable region, the method comprising: (a) Modifying the nucleic acid encoding the polypeptide to alter the charge of at least one exposed amino acid residue on the surface of the polypeptide CDR; (b) culturing host cells to express the said nucleic acid; and (c) Collect the polypeptide containing the antibody variable region from the host cell culture, wherein controlling the pharmacokinetics in plasma refers to prolonging or shortening the half-life of the polypeptide in plasma, wherein a decrease in the isoelectric point of the polypeptide prolongs its half-life in plasma, and an increase in the isoelectric point of the polypeptide shortens its half-life in plasma, wherein the amino acid residues exposed on the CDR surface are at least one amino acid residue selected from amino acid residues at positions 31, 61, 62, 64, and 65 of the Kabat number in the heavy chain variable region and amino acid residues at positions 24, 27, 53, 54, and 55 of the Kabat number in the light chain variable region. The modification involves altering the charge of amino acid residues through amino acid substitution, and the modified amino acids are selected from either group (a) or (b) below: (a) Glutamic acid (E) and aspartic acid (D); and (b) Lysine (K), arginine (R), and histidine (H), The polypeptide containing the antibody variable region further includes an IgG Fc region capable of binding FcRn. 9. The method of claim 8, wherein the polypeptide comprising the antibody variable region is an IgG antibody. 10. The method of claim 8, wherein the polypeptide comprising the antibody variable region is a multispecific polypeptide that binds to at least two types of antigens. 11. The method of claim 8, wherein the polypeptide comprising the antibody variable region is a chimeric antibody, a humanized antibody, or a human antibody. 12. The method of claim 8, wherein the polypeptide prior to modification is a humanized antibody. 13. The method of claim 8, wherein the change in the charge of the amino acid residues results in a change in the theoretical isoelectric point of 1.0 or higher. 14. The method of claim 8, wherein pharmacokinetic control refers to increasing or decreasing any one of the following parameters: plasma clearance CL, area under the concentration curve AUC, mean plasma residence time, and plasma half-life t1/2.